Synthesis of protected 2-pyrrolylalanine for peptide chemistry and examination of its influence on prolyl amide isomer equilibrium

Article abstract of DOI:10.1021/jo3005809

Protected enantiopure 2-pyrrolylalanine was synthesized for application in peptide science as an electron-rich arylalanine (histidine) analog with π-donor capability. (2S)-N-(Boc)-N′-(Phenylsulfonyl)-, (2S)-N,N′-bis-(phenylsulfonyl)-, and (2S)-N,N′-bis-(Boc)-3-(2- pyrrolyl)alanines (10, 3, and 14, respectively) were made in 13-17% overall yields and six to seven steps from oxazolidine β-methyl ester 4. Homoallylic ketone 5 was prepared by a copper-catalyzed cascade addition of vinylmagnesium bromide to ester 4 and converted to pyrrolyl amino alcohol 7 by olefin oxidation and Paal-Knorr condensation. Protecting group shuffle and oxidation of the primary alcohol enabled the synthesis of pyrrolylalanines. The bis-Boc analog 14 proved useful in peptide chemistry and was employed to make N-acetyl-pyrrolylalaninyl-proline N′′-methylamide 25. A study of the influence of the pyrrole moiety on the prolyl amide isomer equilibrium of 25 using ¹H NMR spectroscopy in chloroform, DMSO, and water demonstrated that the pyrrolylalanine peptide exhibited behavior and conformations different from those of other arylalanine analogs.

Full text of DOI:10.1021/jo3005809

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Article
Synthesis of Protected 2-Pyrrolylalanine for Peptide Chemistry and
Examination of its Influence on Prolyl Amide Isomer Equilibrium.
Aurélie A. Dörr, and William D. Lubell
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo3005809 • Publication Date (Web): 14 Jun 2012
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Synthesis of Protected 2-Pyrrolylalanine for Peptide Chemistry and
Examination of its Influence on Prolyl Amide Isomer Equilibrium.
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Aurélie A. Dörr and William D. Lubell*
Département de Chimie, Université de Montréal, C.P.6128. Succursale Centre-Ville.
Montréal, Québec, Canada H3C 3J7
*E-mail : lubell@chimie.umontreal.ca
Table of Contents graphic
Abstract
Protected enantiopure 2-pyrrolylalanine was synthesized for application in peptide
science as an electron rich arylalanine (histidine) analog with π-donor capability. (2S)-N-
(Boc)-N’-(Phenylsulfonyl)-, (2S)-N,N’-bis-(phenylsulfonyl)-, and (2S)-N,N’-bis-(Boc)-3-
(2-pyrrolyl)alanines (10, 3 and 14) were made in 13-17% overall yields and six to seven
steps from oxazolidine β-methyl ester 4. Homoallylic ketone 5 was prepared by copper-
catalyzed cascade addition of vinylmagnesium bromide to ester 4, and converted to
pyrrolyl amino alcohol 7 by olefin oxidation and Paal-Knorr condensation. Protecting
group shuffle and oxidation of the primary alcohol afforded the pyrrolylalanines, among
which bis-Boc analog 14 proved most useful in peptide chemistry to make N-acetyl-
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pyrrolylalaninyl-proline N’-methylamide 25. Study of the influence the pyrrole moiety on
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the prolyl amide isomer equilibrium in 25 using H NMR spectroscopy in chloroform,
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DMSO, and water demonstrated that the pyrrolylalanine peptide exhibited different
behaviour and conformations from those of other arylalanine analogs.
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INTRODUCTION
Structurally diverse amino acid derivatives have widespread use in the physical
and life sciences.1 For example, phenylalanine2a-f and histidine2f-h have been used to study
the importance of π-interactions for peptide folding, recognition and biological activity,4-8
because they possess differing degrees of aromatic-ring electron density.2,3 At the N-
terminus of proline in peptides, Phe may augment the prolyl amide cis-isomer population,
decrease the rate of amide isomerization, and limit protein folding.8-14 Stabilization of the
cis-isomer has been suggested to be due to an electrostatic interaction between the prolyl
amide nitrogen and the Phe aromatic π-system,5a,5f,9 in part because prolyl amide isomer
population has been modulated by varying the electronic density of the side chain of the
residue N-terminal to proline in peptides.15,16,17 For example, in Ac-Xaa-Pro-NHMe, the
cis-amide isomer population was reduced on changing the Xaa residue from Phe to
electron deficient arylalanine residues,17 such as pyradizinylalanine.16
Histidine analogs (i.e., thienylalanine, furylalanine, triazolylalanine,
tetrazolylalanine, thiazolylalanine, imidazolylalanine, and pyrazolylalanine) have been
used to study the importance of the hydrogen bond acceptor and donator properties of the
imidazole side chain.2f-h Components of naturally occurring peptides from marine
organisms,18 such amino acids bearing five member heterocycle side chains have been
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employed in peptidomimetics,19 macromolecular scafolds20 and high throughput
synthesis.21 Although pyrrolylalanine (1,22c Figure 1) represents a promising histidine
surrogate, few syntheses give enantiopure product suitable for peptide chemistry.22,23 For
example, the natural pyrrolylalanine analog from the poisonous mushroom Clitocybe
acromelalga, (2S)-3-(2-carboxy-4-pyrrolyl)alanine (2) has been synthesized in protected
form as a racemate,22f and in optically active form from L-aspartic acid.23a-c
Enantiomerically pure (>94% ee) (2S)-N-Cbz-3-(2-pyrrolyl)alanine derivatives were
synthesized by enantioselective hydrogenation of the corresponding methyl and tert-butyl
(Z)-α,β-didehydroalaninates.23d (2S)-N-Boc-N’-Acetyl-3-(2-pyrrolyl)alanine was also
synthesized from L-aspartic acid by a route featuring ring-closing metathesis and
aromatization to form the heterocycle.23e Prior to our preliminary research,17 to the best of
our knowledge,22,23 pyrrolylalanine has been used only once in the synthesis of a peptide
analog. Racemic N-Cbz-N’-Boc-3-(2-pyrrolyl)alanine was introduced into a thyrotropin-
releasing hormone (TRH, pGlu-His-Pro-NHMe) analog to investigate the role of the
histidine residue; however, no biological activity was reported.22b
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FIGURE 1 Protected pyrrolylalanines analogs.
In the context of research on biologically active peptides bearing histidine
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residues,24,25 we have pursued the development of a effective methodology for the
synthesis of enantiomerically pure 3-(2-pyrrolyl)alanine in a form suitable for
introduction into peptides. Recently, we communicated the synthesis of N,N’-bis-
(phenylsulfonyl)-3-(2-pyrrolyl)alanine (3, Figure 1) by a route featuring a copper-
catalyzed cascade addition of vinyl magnesium bromide to aspartic acid-derived β-amino
ester 4 (Scheme 1).17 Although pyrrolylalanine was obtained in 13% overall yield from 4
and introduced into a dipeptide, attempts were unsuccessful in removing the sulfonyl
groups. Alternative strategies have now been developed for the synthesis of enantiopure
(2S)-3-(2-pyrrolyl)alanine derivatives, which are more suitable for peptide synthesis as
validated by incorporation into Ac-Xaa-Pro-NHMe dipeptide models. The influence of
(2S)-2-pyrrolylalanine on prolyl amide isomer population was subsequently examined by
comparing measurements of cis-isomer populations with related arylalanine derivatives.
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RESULTS AND DISCUSSION
Protected 2-Pyrrolylalanine Synthesis.
Pyrrole 7 was prepared in three steps from β-amino ester 4, which was obtained in
four steps and 47% yield from L-aspartic acid as chiral educt.16 The Cu-catalyzed cascade
addition of excess vinylmagnesium bromide to methyl ester 4 in THF at –45°C gave
homoallylic ketone 5 in 63% yield (Scheme 1).16,26 Pyrrole 7 was prepared by olefin
oxidation and Paal-Knorr condensatrion. γ-Ketoaldehyde 6 was made from olefin 5 using
a mixture OsO4-NaIO4 and 2,6-lutidine in dioxane/water in 95% yield.27 Condensation of
γ-ketoaldehyde 6 with ammonium formate in the presence of a mixture of NaOAc/AcOH
(1:1, 1 eq w/w) in acetonitrile at 65°C gave pyrrole 7 in 72% yield.
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SCHEME 1 Synthesis of N-(Boc)-N’-(Phenylsulfonyl)-3-(2-pyrrolyl)alanine 10.
In earlier research on bipyrrole and prodigiosin analogs,28 sulfonamide protection
of the ring nitrogen prevented destruction of the electron rich pyrrole during double bond
oxidation.29 To oxidize the primaryl alcohol to the carboxylic acid without pyrrole
degradation,30 pyrrole 7 was thus protected as phenysulfonyl counterpart 8 with t-BuOK,
18-c-6 and phenylsulfonyl chloride in THF in 84% yield. Oxazolidine 8 was
subsequently ring opened using 80% aqueous acetic acid at 50°C overnight to afford N-
(Boc)amino alcohol 9 in 81% yield.
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Oxidation of N-(Boc)amino alcohol 9 to (2S)-N-(Boc)-N’-(phenylsulfonyl)-3-(2-
pyrrolyl)alanine 10 proved challenging. Poor yields of acid 10, at best 21-50% from
oxidation of 9 using TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) free radical, sodium
chlorite, and sodium hypochlorite in a sodium-phosphate-buffered acetonitrile
solution,16,31,32 inspired a change in amine protection from Boc to benzenesulfonyl. The
carbamate was removed using 25% trifluoroacetic acid (TFA) in dichloromethane and the
sulfonamide was installed using phenylsulfonyl chloride and Na2CO3 in dioxane/water to
provide (2S)-N,N’-bis-(phenylsulfonyl)-3-(2-pyrrolyl)alaninol 11 in 64% overall yield
(Scheme 2). Using the TEMPO oxidation conditions described above, (2S)-N,N’-bis-
(phenylsulfonyl)-3-(2-pyrrolyl)alanine 3 was obtained from N-(phenylsulfonyl)amino
alcohol 11 in 74% yield. Although 2-pyrrolylalanine targets were achieved because of
sulfonamide protection, low yields in attempts at sulfonyl group removal (i.e., TBAF in
THF, SmI2 in DMPU/THF, and Mg in MeOH) plagued efforts to employ amino acids 3
and 10 in peptide synthesis.17
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SCHEME 2 Synthesis of N,N’-Bis-(Phenylsulfonyl)-3-(2-pyrrolyl)alanine 3.
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To facilitate protecting group removal during peptide synthesis, N,N’-bis-(Boc)-3-
(2-pyrrolyl)alanine 14 was subsequently targeted. Pyrrole 7 was protected with di-tert-
butyl dicarbonate and catalytic 4-(dimethylamino)pyridine (DMAP) in acetonitrile to
afford N-(Boc)pyrrole 12 in 98% yield (Scheme 3).33 Removal of the oxazolidine from 12
with 80% aqueous acetic acid furnished N,N’-bis-(Boc)-3-(2-pyrrolyl)alaninol 13 in 96%
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yield. Most attempts (e.g., TEMPO32, NaIO4-RuCl3,34 H5IO6-CrO3,35 Pt/O2 ) failed
however to convert alcohol 13 to carboxylic acid 14, except for pyridinium dichromate
(PDC) in DMF,37 which provided N,N’-bis-(Boc)-3-(2-pyrrolyl)alanine in 42% yield.
Alternatively, acid 14 was produced by a two-step sequence featuring oxidation to the
aldehyde with oxalyl chloride and DMSO,38 followed by NaClO2 oxidation in 66%
overall yield;39 however, the specific rotation of amino acid 14 was lower [–12.61 versus
–19.14 (c 9.6 10-3, CHCl3)] after the two-step process, suggesting the amino aldehyde
intermediate was configurationally labile.40
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SCHEME 3 Synthesis of N,N’-Bis-(Boc)-3-(2-pyrrolyl)alanine 14.
The enantiomeric purity of N,N’-bis-(Boc)-3-(2-pyrrolyl)alanine 14 was
ascertained after conversion to diastereomeric dipeptides 15 by coupling respectively, to
L- and L,D- serine methyl ester hydrochloride, using N-ethyl N’-(3-dimethylamino
propyl)carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and DIEA
in dichloromethane. Observation of the crude dipeptides 15 and measurement of the
diastereomeric α-proton multiplets of the 3-pyrrolylalanine residue at 4.74 and 4.84 ppm
in 1H NMR spectra in C6D6 during incremental additions of (S,R)-15 in a sample of (S,S)-
15 demonstrated (S,S)-N,N’-bis-(Boc)-3-(2-pyrrolyl)alaninyl-serine methyl ester to be of
a > 98:2 diastereomeric ratio. Hence, (2S)-3-(2-pyrrolyl)alanine 14 is considered to be of
the same high enantiomeric purity.
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In an alternative route, N,N’-bis-(Boc)-3-(2-pyrrolyl)alanine 14 was obtained from
alcohol 16 (Scheme 4). Reduction of the α-carboxylate of β-methyl N-(Boc)aspartate
gave alcohol 16.41 Homoallylic ketone 17 was obtained in 66% yield on treatment of 16
with vinylmagnesium bromide and catalytic CuCN, followed by purification by flash
chromatography.16 Homoallylic ketone 17 (Rf = 0.36, 50:50 ethyl acetate/hexane) existed
in equilibrium with hemiacetal 18 (Rf = 0.77, 50:50 ethyl acetate/hexane). Oxidation of
the mixture of 17 and 18 with NaIO4, followed by condensation of the resulting γ-
ketoaldehyde 19 with ammonium formate delivered pyrrole 20 in 36% yield over two
steps. Although selective pyrrole nitrogen protection of N-(Boc)-3-(2-pyrrolyl)alaninol
20 was unsuccessful, N,N’,O-tris-(Boc)-3-(2-pyrrolyl)alaninol 21 was prepared in 80%
yield by employing excess (Boc)2O and DMAP in acetonitrile. Attempting to cleave
selectively the O-Boc group, treatment of 21 with K2CO3 in MeOH yielded a separable
mixture of N,N’-bis-(Boc)-3-(2-pyrrolyl)alaninol 13 (35-39% yield), N,O-bis-(Boc)-3-(2-
pyrrolyl)alaninol 22, N-(Boc)-3-(2-pyrrolyl)alaninol 20, and starting material 21
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contingent on reaction time.
Recycling compounds 20-22 through
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protection/deprotection process augmented the yield of N,N’-bis-(Boc)-3-(2-
pyrrolyl)alaninol 13.
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SCHEME 4 Synthesis of N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninol 13 from methyl ester 16.
Synthesis and Conformational Analysis of Ac-Xaa-Pro-NHMe
Dipeptide Models.
Steric effects between amino acid side chains favour usually the trans- over the
cis-amide isomer. In the case of amides N-terminal to proline, however, the tertiary
amide may adopt significant amounts of cis-isomer, which are augmented by
phenylalaninyl residues relative to less bulky alaninyl counterparts.9 Stacking of the
aromatic and pyrrolidine rings has been suggested to account for aromatic residues N-
terminal to proline augmenting the prolyl cis-amide population relative to non-aromatic
amino acids in dipeptide models, such as Ac-Xaa-Pro-NHMe and Ac-Xaa-5-tBuPro-
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NHMe.9 Examining the effect of aromatic ring electron density on the magnitude of the
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arylalanine,
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methylpyridazinyl)alanine (Pal) was previously synthesized and incorporated into the
dipeptide model Ac-Xaa-Pro-NHMe, in which Pal reduced significantly the prolyl amide
cis-isomer population relative to Phe.16 Exploring the stacking interaction further, the
placement of the π-enriched arylalanine analog, (2S)-pyrrolylalanine, N-terminal to
proline has now been examined with the expectation to augment the prolyl cis-amide
isomer population by increasing electron density of the π-system. Dipeptide models Ac-
Xaa-Pro-NHMe 25-28 were synthesized to perform head to head comparisons between
arylalanines having different π-density.
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N,N’-bis-Boc-3-(2-pyrrolyl)alanine 14 was coupled to proline N’’-methylamide
hydrochloride using TBTU, HOBT, and DIEA to give N,N’-bis-phenylsulfonyl- and
N,N’-bis-Boc-3-(2-pyrrolyl)alaninyl-proline N’’-methylamides 23 in 91% yield (Scheme
5). Selective removal of the Boc group from the α-amine without loss of the N-Boc-
pyrrole was accomplished by treating dipeptide 23 with 25% TFA in dichloromethane. N-
Acetyl-N’-Boc-3-(2-pyrrolyl)alaninyl-proline N’’-methylamide 24 was produced by N-
acetylation with acetic anhydride and potassium carbonate in dichloromethane in 92%
overall yield from 23. Removal of the Boc group from the pyrrole of 24 was
accomplished by thermolysis on heating at 180°C in 1,2-dichlorobenzene to give N-
acetyl-3-(2-pyrrolyl)alaninyl-proline N’-methylamide 25 in 70% yield.42 For
comparisons, N-acetyl-pyridizinyl-, cyclohexyalaninyl and phenylalaninyl-proline N’-
methylamides 26-28 were prepared as previously described.16
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SCHEME 5 Synthesis of N-(acetyl)dipeptide N’’-Methylamide 25.
The prolyl major trans- and minor cis-amide isomers were analyzed by NMR
spectroscopy in chloroform on the basis of their characteristic nuclear Overhauser effects
between the α-proton of the N-terminal Xaa residue and either the α-proton or the δ-
protons of the proline residue in the NOESY spectra in chloroform.43 The relative
populations of the amide cis- and trans-isomers N-terminal to the prolyl residues of
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dipeptide models 25-28 were ascertained by H NMR spectroscopy in chloroform,
dimethylsulfoxide, and water (Table 1). The percentage of amide cis- and trans-isomer
populations of 25 were measured by integration of the isomeric signals for the 2-
pyrrolylalanine α-proton in the 1H NMR spectra in chloroform and dimethylsulfoxide, in
which the α-proton signal for the trans-isomer appeared downfield from that of the cis-
isomer (Table 2). In water, isomer populations of 25 were measured by integration of the
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N’-methyl doublets in the H NMR spectra. On the other hand, the percentage of amide
cis- and trans-isomer populations of 26 were measured by integration of the isomeric
signals for the pyridazinylalanine α-protons in the 1H NMR spectra in the three solvents.
In the case of the cyclohexylalanine and phenylalanine analogs 27 and 28, isomer
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populations were measured by integration of the N’-methyl doublets in the H NMR
spectra in the three solvents. In addition, the N’’-methylamide and acetamide proton
signals of the major trans-amide conformers of 25-28 were assigned based on their
characteristic coupling patterns as confirmed by TOCSY spectra in chloroform,
dimethylsulfoxide, and water (Table 1).
TABLE 1 Influence of Solvent on Chemical Shifts and Amide Isomer Equilibrium of 25-
28a.
(CDCl3→DMSO)a
(CDCl3→D2O)a
% cis-isomer
(CDCl3)a
43
44
45
46
47
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49
50
51
52
53
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55
56
57
58
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entry
N-terminal
residue
δ NHXaa
Δδ NHXaa
Δδ NHXaa
D2O DMSO CDCl3
NHMe
NHMe
NHMe
(S)-25 L-Pyr
27
25
0
6.39
5.93
1.79
1.53
1.72
1.41
(S)-26 L-Pal
(S)-27 L-Cha
(S)-28 L-Phe
a
23
17
31
12
14
35
0
6.50
6.08
6.24
7.80
6.69
6.36
1.82
1.92
1.99
–0.11
0.98
1.16
1.89
2.04
1.95
0.18
1.05
1.09
21
20
Values are for the major conformer at 5 mM concentration, 25 °C, determined
respectively by 700 and 500 MHz NMR spectroscopy for 25 and 26-28.
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TABLE 2 Chemical Shifts and Integrations of Peaks used in the Conformational
Analysis of 25a.
6
7
8
9
Hα (Pyr)
N’-Me
δ
(I)
Cis-25
Trans-25
Cis-25
Trans-25
Solvents
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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60
4.86 (m)
(1.00)
CDCl3
D2O
-
2.55 (d)
(1.10)
2.60 (d)
(3.00)
4.33 (q)
(0.28)
4.60 (q)
(0.86)
DMSO
a Chemical shifts are in ppm and integration values are given in parentheses.
To distinguish aromatic from hydrophobic interactions, phenylalanine was
compared with cyclohexylalanine. The choice of solvent enhanced or mitigated the
effects of the side chain N-terminal to proline with the pyrrolidine ring. In the relatively
nonpolar solvent chloroform, the Cha and Phe analogs 27 and 28 exhibited essentially the
same effect on the isomer equilibrium. On the other hand, in the relatively polar solvents
DMSO and water, Phe peptide 28 exhibited significantly increased cis-isomer population
relative to Cha peptide 27, which experienced decrease cis-isomer populations relative to
those observed in chloroform. The aromatic effect is thus more pronounced in polar
(DMSO and water) rather than nonpolar (chloroform) solvent. In addition, the placement
of π-deficient pyridazinylalanine N-terminal to proline in 26 gave lower cis-isomer
populations relative to phenylalanine peptide 28 in all three solvents suggesting
diminished π-electron donor-prolyl electron acceptor interaction destabilized the cis-
isomer.16
Although insertion of π-enriched pyrrolylalanine into peptide 25 was expected to
increase the electrostatic interaction, the effects on cis-isomer population were modest at
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best. Relative to pyridazinylalanine peptide 26, pyrrolylalanine peptide 25 increased cis-
isomer population by only 4% in water and 13% in dimethylsulfoxide; however, no
change was seen in chloroform on varying pyridazinylalanine to pyrrolylalanine and only
the trans-isomer was observed. Relative to phenylalanine peptide 28, pyrrolylalanine
peptide 25 exhibited significantly lower cis-isomer populations in all three solvents.
Evidently, the aromatic ring of pyrrolylalanine had a limited effect on the π-electron
donor-prolyl electron acceptor interaction and other factors, such as potential hydrogen
bonding to the proton of the pyrrole ring as well as nitrogen-nitrogen lone pair repulsion,
appear to play greater roles in controlling the isomer equilibrium.
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To try to gain a better understanding of the deviant behaviour of the
pyrrolylalanine peptide 25, the chemical shifts of the amide protons of the major
conformers of peptides 25-28 were compared in the different solvent systems. The
methylamide proton came typically downfield of the acetamide proton in peptides 26-28
in chloroform; however, in the spectrum of pyrrolylalanine peptide 25, the methylamide
proton (5.93 ppm) was upfield relative to the signal for the acetamide proton (6.39
ppm). To distinguish the relative exposures of the amide protons to solvent, the change
in chemical shift of their signals was examined on switching to more polar solvent,
because in studies of cyclic44 and model linear45 peptides, polar solvents which may form
hydrogen bonds cause the signals of exposed amide protons to downfield shift, yet have
limited influence on the chemical displacement of solvent shielded protons. Downfield
shifts of proton signals upon changes in solvent from chloroform to respectively DMSO
and water were significantly less for the methylamide (∆δ = 0.11-1.16 ppm) relative to
the acetamide (∆δ = 1.82-2.04 ppm) for the trans-amide conformer of 26-28. In contrast,
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on switching solvent from chloroform to dimethylsulfoxide and water, respectively, the
downfield shifts for the proton signals were slightly less for the methylamide (∆δ = 1.53
and 1.41 ppm) than the acetamide (∆δ = 1.79 and 1.72 ppm) for pyrrolylalanine peptide
25. The effects of solvent on amide proton chemical shift were in accord with the trans-
amide conformer of peptides 26-28 adopting a preferred β-turn geometry in which the
methylamide is solvent shielded in a hydrogen bond with the acetamide carbonyl. On the
other hand, the solvent effects on the amide proton signals of 25 were inconsistent with
those of peptides 26-28. From the examination of the behaviour of the amide proton
signals, relative to the conformers of peptides 26-28, the conformational preferences of
peptide 25 were different, due evidently to the unique interactions of the pyrrole ring with
the other functionality in the peptide chain.
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26
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CONCLUSION
Effective methods for synthesizing of enantiopure protected 3-(2-pyrrolyl)alanine
analogs have been developed starting from aspartic acid. (2S)-N,N’-Bis-(Boc)-3-(2-
pyrrolyl)alanine 14 (>96% ee) was synthesized in 17% overall yields in six steps from
oxazolidine β-methyl ester 1 and inserted successfully into dipeptide model Ac-Xaa-Pro-
NHMe 25. Conformational analysis of 25 and comparisons with related peptides 26-28
demonstrated that the pyrrole side chain had limited influence on the prolyl cis-amide
isomer populations compared to those of other aromatic residues, such as phenylalanine
in peptide 28, and adopted different conformational preferences. 3-(2-Pyrrolyl)alanine 14
can be introduced into peptides effectively and may be employed as an asparagine or
histidine surrogate. Future applications of 14 to study structure-function relationships in
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peptide science and medicinal chemistry are under investigation and will be reported in
due time.
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17
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23
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25
26
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EXPERIMENTAL SECTION
Materials and Methods. Unless otherwise noted, all reactions were run under a nitrogen
atmosphere and distilled solvents were transferred by syringe. Anhydrous solvent (THF,
CH3CN, DMF and CH2Cl2) were obtained by passage through solvent filtration systems
(GlassContour, Irvine, CA). DIEA was distilled over ninhydrin and CaH2. Final reaction
mixture solutions were dried over MgSO4 or Na2SO4. Chromatography was on 230-400
mesh silica gel, and TLC was on glass-backed silica plates. All compounds were purified
by flash column chromatography on silica gel.46 L-Serine methylester hydrochloride and
D,L-serine methyl ester hydrochlorides were purchased from Aldrich and proline N-
methylamide hydrochloride was synthesized from N-Boc-proline.9 1H NMR spectra were
measured in CDCl3 and C6D6 at 700/400/300 MHz and referenced to CDCl3 (7.26 ppm)
and C6D6 (7.16 ppm). For compound 26, 1H NMR spectra were also measured in CDCl3,
DMSO-d6 and D2O at 700 MHz. 13C NMR spectra were measured in CDCl3 at
175/100/75 MHz and referenced to CDCl3 (77.16 ppm). The chemical shifts for the
carbons and the protons of the minor isomers are respectively reported in parentheses and
in brackets. Mixing times of 800 and 80 ms was used for the NOESY and TOCSY
spectra respectively. Relative populations of prolyl cis- and trans-amide isomers of
compounds 23, 24, and 25 were determined in choroform at 5 mM at 25°C. HRMS
measurements were made on a LC-MSD TOF (Agilent) mass analyzer.
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(S)-4-(1-tert-Butyloxycarbonyl-1H-pyrrol-2-ylmethyl)-2,2-dimethyl-oxazolidine-3-
carboxylic acid tert-butyl ester (12).
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8
9
A stirred solution of pyrrole 7 (prepared according to ref 17, 2.71 g, 9.66 mmol) in
MeCN (140 mL) at room temperature was treated with DMAP (120 mg, 0.98 mmol) and
(Boc)2O (2.57 g, 11.77 mmol) and stirred overnight. The volatiles were removed by
evaporation and the residue was partitioned between H2O (300 mL) and Et2O (300 mL).
The layers were separated and the aqueous phase was extracted with Et2O (2 × 300 mL).
The combined organic extracts were washed with H2O (300 mL) and brine (300 mL),
dried (Na2SO4), filtered and evaporated to give N-(Boc)pyrrole 12 (3.58 g, 98%) as a
sticky yellow oil. TLC Rf 0.37 (10:90 EtOAc/hexane). [α]D20 –17.91 (c 8 × 10–3, CHCl3).
1H NMR (400 MHz, CDCl3) indicated a 1 :1 mixture of carbamate isomers, δ 1.35 (s,
12H), 1.41-1.55 (2 s partially overlapped, 14H), 1.59 (s, 18H), [1.68, (s, 4H)]), 3.09-3.30
(2 m partially overlapped, 4H), 3.81 (d, J = 8.8 Hz, 2H), 3.94 (m, 2H), 4.25-4.39 (2 m
partially overlapped, 2H), 5.85-5.97 (2 br s partially overlapped, 2H), 6.02-6.12 (2 br s
partially overlapped, 2H), 7.19 (s 2H). 13C NMR (100 MHz, CDCl3), δ 152.3, 132.6,
121.2, 113.3 (112.1), 110.5 (110.2), 94.2 (93.5), 83.5, 79.6, 67.7 (67.2), 56.7, 33.3 (32.1),
(28.6) 28.5, 28.3, (27.9) 27.5, (24.8) 23.5. HRMS (ESI) Calcd m/z for C20H32N2NaO5 [(M
+ Na)+] 403.2203, found 403.2221.
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(2S)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninol (13).
Oxazolidine 12 (3.48 g, 9.17 mmol) was dissolved in 180 mL of 80 % aq AcOH (5.6 mL
per 1 mmol oxazolidine). The mixture was stirred at 50°C and monitored by TLC [12 : Rf
0.95 (15:85 EtOAc/dichloromethane) ; 13 : Rf 0.77 (15:85 EtOAc/dichloromethane)].
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After 2.5h, the reaction mixture was cooled to room temperature, concentrated by rotary
evaporation and treated with brine (200 mL). The aqueous layer was extracted with
EtOAc (3 × 300 mL). The combined organic phases were washed with H2O (200 mL),
NaHCO3 (100 mL) and brine (200 mL), dried (MgSO4), filtered and evaporated to a
residue, which was purified by silica gel column chromatography (2 % methanol in
dicloromethane) to give alcohol 13 as a pale yellow viscous oil after lyophilisation (2.99
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20
g, 96 %): TLC Rf 0.22 (3:97 MeOH/dichloromethane). [α]D –21.16 (c 7.6 × 10–3,
1
CHCl3). H NMR (400 MHz, CDCl3), δ 1.41 (s, 9H), 1.6 (s, 9H), 2.93-3.10 (br m, 2H),
3.19 (dd, J = 6.4, 14.8 Hz, 1H), 3.58 (dd, J = 3.6, 10.8 Hz, 1H), 3.65 (br d, J = 10.8 Hz,
1H), 3.86 (m, 1H), 6.06-6.12 (t, J = 3.4 Hz and br s partially overlapped, 2H), 7.17 (dd, J
= 2, 3.2 Hz, 1H).13C NMR (100 MHz, CDCl3), δ 156.5, 150.3, 132.0, 121.6, 114.0,
110.6, 84.3, 79.6, 65.1, 53.7, 29.9, 28.6, 27.9. HRMS (ESI) Calcd m/z for C17H28N2NaO5
[(M + Na)+] 363.1890, found 363.1905.
(2S)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alanine (14) from PDC oxidation.
A stirred solution of alcohol 13 (56.5 mg, 0.166 mmol) in DMF (0.6 mL) at room
temperature was treated with PDC (312 mg, 0.830 mmol), stirred for 24 h, and
partitioned between H2O (5 mL) and ethyl acetate (5 mL). The mixture was extracted
with ethyl acetate (3 × 3 mL). The combined organic phases were washed with H2O (5
mL) and brine (5 mL), dried (MgSO4), filtered and evaporated to give a residue, which
was purified by silica gel column chromatography (2-5 % methanol in dichloromethane)
to give acid 14 as a white foam (24.2 mg, 41 %): TLC Rf 0.22 (3:97
20
1
MeOH/dichloromethane). [α]D –21.16 (c 7.6 × 10–3, CHCl3). H NMR (400 MHz,
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CDCl3), δ 1.20-1.50 (2 s, 9H), 1.61 (s, 9H), 3.20-3.33 (m, 1H), 3.50-3.68 (m, 1H), 4.55
(br s, 1H), 5.36 (br d, J = 6.4 Hz, 1H), 6.09 (d, J = 2.8 Hz, 1H), 7.22 (s, 1H), 8.66 (br s,
7
8
9
13
0.4H). C NMR (100 MHz, CDCl3), δ 156.1, 150.0, 148.4, 130.3, 122.2, 114.4, 110.5,
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84.4, 80.4, 54.2, 30.9, 28.5, 28.2. HRMS (ESI) Calcd m/z for C17H26N2NaO6 [(M + Na)+]
377.1683, found 377.1702.
(2S)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alanine (14) by way of aldehyde intermediate.
A solution of oxalyl chloride (50 µL, 0.565 mmol) in CHCl2 (1 mL) at –78°C was
treated slowly with a solution of DMSO (48 µL, 0.678 mmol) in CHCl2 (1 mL), stirred
for 10 min, treated with a solution of alcohol 13 (38.5 mg, 0.113 mmol) in CHCl2 (2 mL),
stirred for 30 min, treated with DIEA (197 µL, 113 mmol), allowed to warm to 0°C with
stirring for 10 min, and quenched with brine (4 mL). The reaction mixture was extracted
with in CHCl2 (3 × 10 mL). The combined organic phases were dried (MgSO4), filtered
and concentrated under reduced pressure. Without further purification, the aldehyde
intermediate was used immediately in the next step.
The aldehyde in t-BuOH (3 mL) at room temperature was treated with 2-methyl-2-butene
(96 µL, 0.904 mmol) in THF (9 mL), followed by drop-wise addition of a solution of
sodium chlorite (34 mg, 0.339 mmol) and potassium dihydrogenphosphate (42 mg, 0.339
mmol) in H2O (9 mL). The mixture was stirred vigorously for 22 h at room temperature,
partitioned between saturated aqueous NH4Cl (45 mL) and ethyl acetate (45 mL), and the
aqueous phase was extracted with ethyl acetate (3 × 30 mL). The combined organic
phases were washed with brine (30 mL), dried (MgSO4), filtered, and concentrated to
give a residue, which was purified by column chromatography (1-5% MeOH in
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dichoromethane) to provide 2-pyrrolylalanine 14 (26.6 mg, 66%) as a white foam
exhibiting identical spectroscopic characteristics as described above: [α]D20 –12.61 (c 9.6
× 10–3, CHCl3).
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(S,S)- and (S,RS)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninyl-serine methyl ester (15).
A solution of 2-pyrrolylalanine 14 (30 mg, 0.085 mmol), HOBt (11.5 mg, 0.085 mmol),
DIEA ( 44.5 µL, 0.085 mmol), and L-serine ester methyl hydrochloride (26.5 mg, 0.170
mmol) in CH2Cl2 (3.5 mL) was stirred at 0°C for 15 min, treated with EDCI (13.3 mg,
0.085 mmol), and allowed to warm to room temperature. After stirring for 2.5 h, TLC
showed that acid 14 [Rf 0.23 (5:95 EtOAc/dichloromethane)] was completely consumed
and a new less polar product, (S,S)-dipeptide 15 was formed [Rf 0.29 (5:95
EtOAc/dichloromethane)]. The volatiles were evaporated under vacuum and the residue
was dissolved in EtOAc (15 mL) and washed with saturated aqueous NaHCO3 (2 × 5
mL), H2O (5 mL), 1N HCl (2 × 5 mL), H2O (5 mL) and brine (5 mL), dried (MgSO4),
1
filtered and evaporated to give a residue that was examined by 400 MHz H NMR
spectroscopy in C6D6. (S,S)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninyl-serine methyl ester
(S,S)-15: 1H NMR (400 MHz, C6D6) δ 1.30-1.35 (2 s partially overlapped, 18H), 2.93 (br
s, 1H), 3.14 (dd, J = 10.8, 14 Hz, 1H), 3.23 (s, 3H), 3.70 (dd, J = 2.6, 14.6 Hz, 1H), 3.82
(dd, J = 2.2, 11.2 Hz, 1H), 3.95 (br d, J = 3.95 Hz, 1H), 4.64 (m, 1H), 4.74 (m, 1H), 5.46
(br d, J = 7.2 Hz, 1H), 5.98 (2 s partially overlapped, 2H), 7.21 (br t, J = 2.2 Hz, 1H),
7.32 (br d, J = 6.8 Hz, 1H).
(S,RS)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninyl-serine methyl ester (S,RS)-15 was prepared
employing the same protocol except for the use of D,L-serine methyl ester: 1H NMR (400
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MHz, C6D6) δ 1.35 (s, 18H), 1.37 (s, 18H), 2.49 (br s, 1H), 2.67 (br s, 1H), 3.06-3.20
(m, 2H), 3.25 (s, 6H), 3.63-3.76 (m, 2H), 3.76-3.86 (m, 2H), 3.88-4.01 (m, 2H), 4.60 (m,
1H), 4.64 (m, 1H), 4.74 (m, 1H), 4.84 (m, 1H), 5.37 (br d, J = 6.4 Hz, 1H), 5.44 (br d, J =
6.4 Hz, 1H), 5.95-6.03 (m, 4H), 7.25 (s, 4H).
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The measurement of the limits of detection of (S,R)-15 in a sample of (S,S)-15 was
performed by integration of the signals of the diastereomeric α-proton multiplets at 4.74
1
and 4.84 ppm in H NMR spectra in C6D6 during incremental additions of (S,RS)-15,
which demonstrated (S,S)-N,N’-bis-(Boc)-3-(2-pyrrolyl)alaninyl-serine methyl ester
(S,S)-15 to be of >96% diastereomeric purity. Hence, (2S)-3-(2-pyrrolyl)alanine 14 is
considered to be of the same high enantiomeric purity.
(2S)-tert-butyl (1-hydroxy-4-oxo-oct-7-en-2-yl)carbamate (17) and tert-butyl ((3S)-5-
(but-3-en-1-yl)-5-hydroxytetrahydrofuran-3-yl) carbamate (18).
In a flame dried flask, under argon, CuCN (797 mg, 8,95 mmol) was treated with freshly
prepared 1M vinylmagnesium bromide in THF (96 mmol, 96 mL) at –45°C, stirred for 30
min, treated via syringe over 10 min with a solution of methyl ester 16 (3.48 g, 14.92
mmol) in THF (30 mL), and stirred at –45°C for 1 h. The cold bath was removed and
replaced with an ice bath and the mixture was stirred overnight during which time the
bath had warmed to room temperature. The mixture was cooled to 0 °C, quenched with
1M aqueous NaH2PO4 (250 mL) and shaken vigorously. The layers were separated. The
aqueous phase was extracted with Et2O (3 × 250 mL). The combined organic extracts
were washed with saturated NaHCO3 solution (250 mL) and brine (250 mL), dried
(MgSO4), filtered and evaporated to a residue, which was purified by silica gel column
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chromatography (30-50 % ethyl acétate in hexane) to give acetal 18 as a yellow
visqueous oil (695 mg, 18%), TLC Rf 0.77 (50 :50 EtOAc/hexane) ; and homoallylic
ketone 17 as a white solid (1,84 g, 48%), TLC Rf 0.36 (50 :50 EtOAc/hexane).
Homoallylic ketones 17 and 18 were combined for the next reaction (2.53 g, 66%). In
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1
solution 17 and 18 equilibrated and exhibited the following spectral data: H NMR (300
MHz, CD3Cl3), δ 1.43 (1 s, 9H), 2,32 (q, J = 6.67, 14.12 Hz, 2H), 2.55 (t, J = 7.30 Hz,
2H), 2.76 (d, J = 5.85 Hz, 2H), 3.67 (t, J = 5.10 Hz, 2H), 3.95 (m, 1H), 4.96-5.06 (m,
2H), 5.26 (br s, 1H), 5.79 (m, 1H).
(2S)-N-(Boc)-3-(2-pyrrolyl)alaninol (20).
2,6-Lutidine (2.18 mL, 18.76 mmol), OsO4 (2.5% in tert-butanol, 235.5 µL, 0.188 mmol)
and NaIO4 (8.02 g, 37.52 mmol) were added to a solution containing a mixture of
homoallylic ketone 17 and acetal 18 (2.41g, 9.38 mmol) in 3:1 dioxane/water (94 mL).
The mixture was stirred at room temperature for 18 h, and partitioned between H2O (100
mL) and CH2Cl2 (200 ml). The the aqueous phase was extracted with CH2Cl2 (3 × 100
mL). The combined organic layers were washed with brine (100 mL), dried (Na2SO4),
filtered and evaporated to give aldehyde 19: TLC Rf 0.57 (20:80 EtOAc/hexane). Without
further purification, ketoaldehyde 19 (2,70 g crude) was treated with NH4CO2H (3,28 g,
52.05 mmol), and NaOAc/AcOH (2.70 g, 100 mol% w/w, prepared by mixing equimolar
quantities of NaOAc and HOAc) in MeCN (260 mL), and heated at 65°C until complete
consumption of starting ketoaldehye 19 was observed by TLC [19 : Rf 0.50 (5:95
EtOAc/chloroform)] and a new less polar product was formed, 20 [Rf 0.57 (80:20 EtOAc
/hexane)]. After 3 h, the mixture was cooled to room temperature, and partitioned
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between pH 6.8 sodium phosphate buffer (200 mL) and a 2:1 Et2O/CH2Cl2 solution (200
mL). The aqueous phase was extracted with 2:1 Et2O/CH2Cl2 (2 × 200 mL). The
combined organic layers were washed with brine (250 mL), dried (MgSO4), filtered and
evaporated to a residue, which was purified by silica gel column chromatography (40-80
% ethyl acetate in hexane) to give pyrrole 20 (813 mg, 36 %) as a pale orange crystalline
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solid: mp 112-114°C; TLC Rf 0.65 (80:20 EtOAc/hexane); [α]D –14.46 (c 14.3 × 10–3,
CHCl3). 1H NMR (400 MHz, CDCl3), δ 1.45 (s, 9H), 2.88 (d, J = 6.4 Hz, 2H), 3.62 (d, J
= 4.8 Hz, 2H), 3.81 (m, 1H), 4.93 (br d, J = 7.6 Hz, 1H), 5.98 (br s, 1H), 6.13 (dd, J =
2.8, 5.6 Hz, 1H), 6.70 (dd, J = 2.8, 4 Hz, 1H), 8.69 (br s, 1H). 13C NMR (100 MHz,
CDCl3), δ 156.5, 127.8, 117.5, 108.5, 107.2, 80.2, 64.4, 52.5, 29.9, 28.6. HRMS (ESI)
Calcd m/z for C12H20N2NaO3 [(M + Na)+] 263.1366, found 263.1367.
(2S)-N,N’,O,tris-(Boc)-3-(2-pyrrolyl)alaninol (21).
A solution of pyrrolylalaninol 20 (766.3 mg, 3.19 mmol) in MeCN (40 mL) at room
temperature was treated with DMAP (47.8 mg mg, 0.306 mmol) and (Boc)2O (1.34 g,
6.12 mmol), stirred overnight, and evaporated to a residue, which was partitioned
between H2O (100 mL) and EtOAc (100 mL). The aqueous phase was extracted with
EtOAc (2 × 100 mL). The combined organic layers were washed with H2O (100 mL) and
brine (100 mL), dried (Na2SO4), filtered and evaporated to give carbonate 21 (1.43 g, 80
20
%) as a pale pink solid: mp 108-110°C ; TLC Rf 0.74 (40:60 EtOAc/hexane); [α]D
–
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22.14 (c 16.5 × 10–3, CHCl3). H NMR (400 MHz, CDCl3) δ 1.37 (s, 9H), 1.50 (s, 9H),
1.60 (s, 9H), 3.00-3.11 (m, 1H), 3.16 (dd, J = 4.6, 15 Hz, 1H), 4.08-4.21 (m, 3H), 4.86
(br d, J = 10.5 Hz, 1H), 6.04 (br s, 1H), 6.07 (t, J = 3.2 Hz, 1H), 7.19 (dd, J = 1.8, 3.4 Hz,
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1H). 13C NMR (100 MHz, CDCl3), δ 155.4, 153.7, 149.9, 131.5, 121.8, 113.7, 110.4,
83.9, HRMS (ESI) Calcd m/z for C22H36N2NaO7 [(M + Na)+] 463.2415, found 463.2429.
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(2S)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninol (13) from carbonate (21).
A solution of carbonate 21 (74 mg, 0.167 mmol) and K2CO3 (69 mg, 0.500 mmol) in
MeOH (5 mL) was stirred at room temperature for 9 h. A solution of saturated NaHCO3
(6 mL) was added to the mixture, which was extracted with EtOAc (3 × 12 mL). The
combined organic layers were washed with brine (10 mL), dried (Na2SO4), filtered, and
evaporated to give a residue, which was separated by column chromatography on silica
gel (20-50 % EtOAc in hexane). First to elute was carbonate 21 (9.8 mg), as a pale pink
solid exhibiting identical characteristics as described above: TLC Rf 0.74 (40:60
EtOAc/hexane). Next to elute was (2S)-N,O-bis-(Boc)-3-(2-pyrrolyl)alaninol 22 (7.2 mg,
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13 %) as a pale yellow solid: mp 79-81°C; TLC Rf 0.58 (40:60 EtOAc/hexane); [α]D
–
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9.13 (c 10.4 × 10–3, CHCl3). H NMR (400 MHz, CDCl3) δ 1.44 (s, 9H), 1.52 (s, 9H),
2.86 (m, 2H), 3.95-4.09 (2 m partially overloped, 2H), 4.09-4.17 (m, 1H), 4.81 (br s, 1H),
5.99 (br s, 1H), 6.13 (dd, J = 2.8, 6 Hz, 1H), 6.72 (dd, J = 2.6, 3.8 Hz, 1H), 8.67 (br s,
1H). 13C NMR (100 MHz, CDCl3), δ 155.6, 154.0, 127.2, 117.7, 108.7, 107.8, 83.1, 80,1,
67,3, 49.9, 29.9, 28.6, 27.9. HRMS (ESI) Calcd m/z for C17H29N2O5 [(M + H)+]
341.2071, found 341.2087; calcd m/z for C17H28N2NaO5 [(M + Na)+] 363.1890, found
363.1908. Next to elute was bis-Boc-pyrrolyl)alaninol 13 (22.5 mg, 39 %) as a pale
yellow viscous oil [TLC Rf 0.26 (40:60 EtOAc/hexane)], followed by N-Boc-
pyrrolylalaninol 20 (12 mg, 30 %) as a pale orange cristalline solid [TLC Rf 0.13 (40:60
EtOAc/hexane)], both exhibiting identical characteristics as described above.
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(S,S)-N,N’-Bis-(Boc)-3-(2-pyrrolyl)alaninyl-proline N’’-methylamide (23).
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A solution of amino acid 14 (50 mg, 0.141 mmol), HOBt (19.1 mg, 0.141 mg), DIEA (74
µL, 0.423 mmol), and proline N’’-methylamide hydrochloride (46.4 mg, 0.282 mmol) in
CH2Cl2 (7 mL) was stirred at 0°C for 15 min, and treated with TBTU (42.3 mg, 0.141
mmol). The ice bath was removed and the mixture was allowed to warm to room
temperature with stirring. After 5 h, the volatiles were evaporated under vacuum. The
residue was dissolved in EtOAc (20 mL), washed with saturated aqueous NaHCO3 (2 × 7
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mL), H2O (5 mL), 1N HCl (2 × 7 mL), H2O (7 mL) and brine (7 mL), dried (MgSO4),
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filtered and evaporated to give dipeptide 23 as a white residue (59.3 mg, 91 %): [α]D
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36.82 (c 16.3 × 10–3, CHCl3). H NMR (700 MHz, CDCl3) indicated a 30:70 ratio of
amide cis- and trans-isomers N-terminal to proline: δ 1.37 (s, 6 H) [1.39 (s, 2 H)], [1.59
(s, 3.6 H)] 1.60 (s, 6.4 H), 1.78-1.87 (m, 1.3 H), 1.92-1.98 ( m, 1.4 H), 2.35-240 (m, 0.7
H) [2.45-2.48 (m, .3H)], 2.72-2.77 (2 d partially overlapped, 3 H), 3.10-3.22 (m, 1.2 H),
3.26 ( dd, J = 6.7, 14.5 Hz, 0.8H), 3.40-3.45 (m, 0.6 H), 3.50-3.54 (m, 0.6 H), 3.72 (q, J =
8.8, 17.7 Hz, 0.7 H), [4.25 (d, J = 7 Hz, 0.3 H)] 4.6 (d, J = 7.8 Hz, 0.7 H), [4.53 (q, J =
6.3, 14 Hz, 0.3 H)] 4.80 (q, J = 7.3, 15.5 Hz, 0.6 H), [5.12 (d, J = 5.7 Hz, 0.3 H)] 5.20 (d,
J = 8.9 Hz, 0.6 H), 6.02 (s, 0.7 H), 6.04-6.09 (m, 1.3 H), 6.67 (br s, 0.6 H) [7.64 (br s, 0.2
13
H)], 7.18-7.22 (2m partially overlapped, 1 H). C NMR (175 MHz, CDCl3), δ 172.8,
171,5, (156.0) 155.3, 149.6, 130.0 (129.2), (122.3) 122.2, (115.0) 114.3, (110.4) 110.2,
(84.7) 84.4, (80.2) 79.8, (60.7) 60.1, (52.5) 51.7, 47.3 (46.9), 32.3, (31.1) 31.0, 28.5
(28.4), 28.1, (27.3) 26.3, 25.1 (22.2). HRMS (ESI) Calcd m/z for C23H37N4O6 [(M + H)+]
465.2708, found 465.2723; calcd m/z for C23H36N4NaO6 [(M + Na)+] 487.2527, found
487.2537.
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(S,S)-N-Acetyl-N’-(Boc)-3-(2-pyrrolyl)alaninyl-proline N’’-methylamide (24).
A solution of N,N’-bis-Boc-dipeptide N’’-methyl amide 23 (24.1 mg, 0.052 mmol) in 1:3
TFA/CH2Cl2 (3 mL) was stirred at room temperature for 30 min and evaporated on a
rotary evaporator. The resulting dipeptide N’’-methyl amide trifluoroacetate was
dissolved in CH2Cl2 (3 mL), treated with K2CO3 (71.9 mg, 0.520 mmol) and Ac2O (49
µL, 0.520 mmol), stirred for 18 h, filtered, washed with CH2Cl2 (2 x 10 mL), and
evaporated to give N-acetyl-N’-Boc-dipeptide N’’-methyl amide 24 (19.3 mg, 92 %) as a
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colorless residue: TLC Rf 0.13 (5:95 MeOH/dichloromethane); [α]D –22.33 (c 15.5 ×
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10–3, CHCl3). H NMR (700 MHz, CDCl3) indicated a 35:65 ratio of amide cis- and
trans-isomers N-terminal to proline, δ [1.58 (s, 3 H)], 1.60 (s, 6 H), 1.81-1.88 (m, 1.5 H),
1.91 (s, 2 H) [1.94 (s, 1 H)], 1.95-2.02 (m, 1.5 H), 2.34-2.38 (m, 0.7 H), 2.47-2.52 (m, 0.3
H), 2.75 (d, J = 4.9 Hz, 2 H) [2.77 (d, J = 4.2 Hz, 1 H)], 3.11-3.17 (m, 1 H), [3.25 (dd, J
= 5.6, 14 Hz, 0.3 H)] 3.33 (dd, J = 5.9, 14.3 Hz, 0.7 H), 3.48-3.57 (m, 1.3 H), 3.80 (m,
0.7H), [4.34 (d, J = 7.7 Hz, 0.3 H] 4.56 (dd, J = 2.1, 8.4 Hz, 0.7 H), [4.52 (m, 0.3 H)]
0.508 (m, 0.7 H), 6.0 (m, 0.6 H), 6.05-6.09 (m, 1.4 H), 6.26-6.31 (2 br d partially
overlapped, 1 H), 6.58 (br d, J = 4.2 Hz, 0.7 H) [7.73 (br d, J = 4.9 Hz, 0.3 H ), 7.18-7.20
(2 m partially overlapped, 1 H).13C NMR (175 MHz, CDCl3), δ (172.4) 171.4, 171.0
(170.9), (170.8) 169.8, (149.7) 149.6, 129.9 (129.2), (122.4) 122.3, (115.0) 114.4, (110.5)
110.1, (84.8) 84.4, (60.7) 60.2, (52.2) 50.5, 47.4 (46.9), 31.9 (31.1), (30.5) 29.8, (28.1)
28.0, (27.4) 26.3, (26.6) 25.1, 23.1 (22.9). HRMS (ESI) Calcd m/z for C20H31N4O5 [(M +
H)+] 407.2289, found 407.2308; calcd m/z for C20H30N4NaO5 [(M + Na)+] 429.2108,
found 429.2124.
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N-Acetyl-3-(2-pyrrolyl)alaninyl-proline N’’-methylamide (25).
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N-Acetyl-N’-Boc-dipeptide N’’-methyl amide 24 (3.8 mg, 0.009 mmol) in 1,2-
dichlorobenzene was heated at 180 °C for 30 min. The mixture was cooled to room
temperature and purified by silica gel column chromatography (3-5 % MeOH in
dichloromethane) to give N-acetyl- dipeptide N’’-methyl amide 25 as a yellow residue
(1.9 mg, 66 %): TLC Rf 0.16 (5:95 MeOH/dichloromethane); [α]D20 –32.22 (c 5.33 × 10–
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3, CHCl3). H NMR (400 MHz, CDCl3) indicated only the trans-isomer: δ 1.81-2.17 (m,
7 H), 2.86 (d, J = 4.8 Hz, 3 H), 2.96-3.07 (m, 2 H), 3.14 (dd, J = 3.4, 14.6 Hz, 1 H), 3.54-
3.62 (m, 1 H), 4.31 (dd, J = 5.4, 8.2 Hz, 1 H), 4.86 (m, 1 H), 6.00 (m, 1 H), 6.06-6.16 (2
m overlapped, 2 H), 3.78 (br d, J = 8 Hz, 1 H), 6.72 (m, 1 H), 9.91 (br s, 1 H). 13C NMR
(100 MHz, CDCl3), δ 173.2, 171.0, 169.7, 125.6, 118.4, 108.3, 108.0, 61.0, 51.7, 47,5,
31.5, 29.7, 26.7, 25.5, 23.5. HRMS (ESI) Calcd m/z for for C15H22N4NaO3 [(M + Na)+]
329.1584, found 329.1591.
ACKNOWLEDGEMENTS
This work was supported by the Natural Sciences and Engineering Research Council of
Canada (NSERC), and Fond Québécois de la Recherche sur la Nature et les Technologies
(FQRNT). The authors would like to thank Dr. Alexandra Fürtös of the Université de
Montréal Mass Spectrometry facility for mass spectral analyses, and Dr. Phan viet Minh
Tan and Dr. Cédric Malveau of the Regional High-Field NMR Laboratory for their
assistance in running 2D NOESY/TOCSY experiments.
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Supportin Information: H and 13C NMR, NOESY, TOCSY and mass spectra of
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new compounds. This material is available free of charge via the Internet at
http://pubs.acs.org.
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