Synthesis and conformational studies of α/β2,3-peptides derived from alternating β2,3-amino acids...
15
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem. Cite this: DOI: 10.1039/c4nj02031f Synthesis and conformational studies of a/b 2,3 -peptides derived from alternating b 2,3 -amino acids and L-Ala repeats† Gangavaram V. M. Sharma,* a Tailor Sridhar,‡ a Bacchu Veena,‡ a Pothula Purushotham Reddy,‡ b Sheri Venkata Reddy, a Christian Bruneau c and Ajit C. Kunwar* b Cyclic b 2,3 -amino acids though have been extensively used in foldamer designs, their acyclic analogues have received less attention. In view of strong backbone constraints imparted by the substituents, b 2,3 -amino acids provide very attractive options for creating novel foldamers. In the present study, new C-linked carbo-b 2,3 -amino acids (b 2,3 -Caa) were prepared by the alkylation of Ca-carbon (C2) with allyl and propargyl halides, and used with L-Ala to design regular 1 : 1 hybrid a/b 2,3 -peptides. Extensive NMR and MD studies revealed the presence of right-handed 11/9-mixed helices in the peptides with the ‘a-b-a’ sequence at the C-terminus, while, induction of ‘turns’ in the peptides with the ‘b-a-b’ motif at the C-terminus. Despite the strong backbone constraints due to the substitution at both the b 2 and b 3 -carbons, the folds of these a/b 2,3 -peptides are less robust compared to foldamers of the a/b 3 -family, reflecting the impact of Ca-substitution. Introduction Designing of oligomers consisting of unnatural amino acids that mimic the natural peptides is an active area of research. 1 b-Peptides, derived from the b-amino acids, were among the first to be investigated for their capability to fold in a well defined and predictable manner to produce ‘foldamers’. 2 b-Amino acids, with an additional C–C bond in the backbone, provide a variety of options for the side chain substitutions, allowing manipulation of the backbone dihedral angles, leading to diversity in structures. The earliest studies on foldamers in b-peptides were from cyclic b 2,3 -monomers 3 which impart very strong backbone dihedral angle constraints, while the studies on peptides with acyclic b 2,3 -residues are rather sparse. Seebach et al. 4 utilized like b 2,3 -amino acid 5 residues in peptide design to generate a 14-helix, while, the oligomers 6 derived from unlike b 2,3 -amino acid residues produced parallel pleated sheets and hairpin arrangements with 14-atom H-bonding. Further, Seebach et al. 7 utilized b 2,3 -amino acids in different designs to ascertain the influence of additional substitution at the Ca-position. The same group also reported 8 a 8-helix in oligo- mers derived from b 2,3 -amino acid with a hydroxy group in the Ca-position. Grierson et al. 9 utilized b 2,3 -amino acid with a hydroxy group in the Ca-position in the peptide design, wherein, they realized a b-strand type structure. Gellman et al. 10 reported the formation of stable reverse turn that promotes the polar anti- parallel sheets in peptides having b 2,3 -amino acids. Since the appearance of the first reports about a decade back on a/b-peptides 11,12 with heterogeneous backbones, the domain of peptidic foldamers has expanded several times and their studies remain a subject matter of considerable contemporary interest. b 2,3 -Amino acids, cyclic and acyclic, though have been utilized in the design of peptides with diverse structures, the first report on the synthesis of 1 : 1 a/b 2,3 -peptides, from acyclic b 2,3 -amino acid and Aib, appeared in 2012 from Muraleedharan et al., 13 wherein, they demonstrated the remarkable ability of the anti-b 2,3 -amino acid to shift to the gauche conformation, for the formation of 11-atom H-bonding. In our studies on the a/b-peptides 11c,14 from C-linked carbo b-amino acids (b 3 -Caa) 15 with a carbohydrate side chain at the Cb-position, the resultant 11/9-helix 16 and other secondary and tertiary structures were generated based on the concept of ‘alternating chirality’. In a further study, to understand the a Organic and Biomolecular Chemistry Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail: [email protected] b Centre for Nuclear Magnetic Resonance and Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India. E-mail: [email protected] c UMR6226: Institut des Sciences Chimiques de Rennes, Universite ´ de Rennes 1, France † Electronic supplementary information (ESI) available: NMR spectra, solvent titration plots, and distance constraints used in MD calculations. See DOI: 10.1039/c4nj02031f ‡ These authors have equally contributed to this work. Received (in Montpellier, France) 13th November 2014, Accepted 22nd January 2015 DOI: 10.1039/c4nj02031f www.rsc.org/njc NJC PAPER Published on 23 January 2015. Downloaded by Hebrew University of Jerusalem on 03/02/2015 17:53:41. View Article Online View Journal
Transcript of Synthesis and conformational studies of α/β2,3-peptides derived from alternating β2,3-amino acids...
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
Cite this:DOI: 10.1039/c4nj02031f
Synthesis and conformational studies ofa/b2,3-peptides derived from alternatingb2,3-amino acids and L-Ala repeats†
Gangavaram V. M. Sharma,*a Tailor Sridhar,‡a Bacchu Veena,‡a
Pothula Purushotham Reddy,‡b Sheri Venkata Reddy,a Christian Bruneauc andAjit C. Kunwar*b
Cyclic b2,3-amino acids though have been extensively used in foldamer designs, their acyclic analogues
have received less attention. In view of strong backbone constraints imparted by the substituents,
b2,3-amino acids provide very attractive options for creating novel foldamers. In the present study, new
C-linked carbo-b2,3-amino acids (b2,3-Caa) were prepared by the alkylation of Ca-carbon (C2) with allyl
and propargyl halides, and used with L-Ala to design regular 1 : 1 hybrid a/b2,3-peptides. Extensive
NMR and MD studies revealed the presence of right-handed 11/9-mixed helices in the peptides with the
‘a-b-a’ sequence at the C-terminus, while, induction of ‘turns’ in the peptides with the ‘b-a-b’ motif at
the C-terminus. Despite the strong backbone constraints due to the substitution at both the b2 and
b3-carbons, the folds of these a/b2,3-peptides are less robust compared to foldamers of the a/b3-family,
reflecting the impact of Ca-substitution.
Introduction
Designing of oligomers consisting of unnatural amino acidsthat mimic the natural peptides is an active area of research.1
b-Peptides, derived from the b-amino acids, were among thefirst to be investigated for their capability to fold in a welldefined and predictable manner to produce ‘foldamers’.2
b-Amino acids, with an additional C–C bond in the backbone,provide a variety of options for the side chain substitutions,allowing manipulation of the backbone dihedral angles, leadingto diversity in structures. The earliest studies on foldamers inb-peptides were from cyclic b2,3-monomers3 which impart verystrong backbone dihedral angle constraints, while the studies onpeptides with acyclic b2,3-residues are rather sparse. Seebachet al.4 utilized like b2,3-amino acid5 residues in peptide design togenerate a 14-helix, while, the oligomers6 derived from unlike
b2,3-amino acid residues produced parallel pleated sheetsand hairpin arrangements with 14-atom H-bonding. Further,Seebach et al.7 utilized b2,3-amino acids in different designsto ascertain the influence of additional substitution at theCa-position. The same group also reported8 a 8-helix in oligo-mers derived from b2,3-amino acid with a hydroxy group in theCa-position. Grierson et al.9 utilized b2,3-amino acid with ahydroxy group in the Ca-position in the peptide design, wherein,they realized a b-strand type structure. Gellman et al.10 reportedthe formation of stable reverse turn that promotes the polar anti-parallel sheets in peptides having b2,3-amino acids.
Since the appearance of the first reports about a decade backon a/b-peptides11,12 with heterogeneous backbones, the domain ofpeptidic foldamers has expanded several times and their studiesremain a subject matter of considerable contemporary interest.
b2,3-Amino acids, cyclic and acyclic, though have beenutilized in the design of peptides with diverse structures, thefirst report on the synthesis of 1 : 1 a/b2,3-peptides, from acyclicb2,3-amino acid and Aib, appeared in 2012 from Muraleedharanet al.,13 wherein, they demonstrated the remarkable ability ofthe anti-b2,3-amino acid to shift to the gauche conformation, forthe formation of 11-atom H-bonding.
In our studies on the a/b-peptides11c,14 from C-linked carbob-amino acids (b3-Caa)15 with a carbohydrate side chain at theCb-position, the resultant 11/9-helix16 and other secondaryand tertiary structures were generated based on the conceptof ‘alternating chirality’. In a further study, to understand the
a Organic and Biomolecular Chemistry Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India. E-mail: [email protected] Centre for Nuclear Magnetic Resonance and Structural Chemistry,
CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India.
E-mail: [email protected] UMR6226: Institut des Sciences Chimiques de Rennes, Universite de Rennes 1,
France
† Electronic supplementary information (ESI) available: NMR spectra, solventtitration plots, and distance constraints used in MD calculations. See DOI:10.1039/c4nj02031f‡ These authors have equally contributed to this work.
Received (in Montpellier, France)13th November 2014,Accepted 22nd January 2015
DOI: 10.1039/c4nj02031f
www.rsc.org/njc
NJC
PAPER
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article OnlineView Journal
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
impact of substitution at the Ca-position on the a/b-peptides,two monomers, cis C-linked carbo b2,3-amino acids (b2,3-Caas)
117 and 2 (Fig. 1), with C-allyl18 and C-propargyl side chainsrespectively, were utilized in the peptide design. The Ca-allyl
Fig. 1 Structures of amino acids 1–3 and their peptides 4–17.
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
b2,3-Caa 1 was utilized for the synthesis of cyclic amides andpeptides via RCM reaction, by our group earlier.17 Thus, theobjective on the use of the b2,3-Caas 1 and 2 in the peptide designis multi-fold, wherein, the allylic and propargylic side chainscontaining sp2 and sp carbons respectively, are not only expectedto show a different impact on the conformations, but also can beused appropriately for the introduction of constraints and func-tional modifications. Herein, the study describes the synthesis ofa/b2,3-peptides 6–17 (Fig. 1) from L-Ala and b2,3-Caas 1 and 2 with1 : 1 alternation, and their conformational analysis by NMR andmolecular dynamics (MD) studies.
Results and discussionSynthesis of monomer 2
Ester 2 was prepared by the adoption of the procedure reported forthe synthesis of monomer 1 by our group earlier.17 Accordingly,reaction of the known15 b3-Caa 2a with propargyl bromide in thepresence of n-BuLi at �78 1C for 2 h gave the ester 2 in 42% yield(Scheme 1). To ascertain the absolute stereochemistry at the C2stereocentre, 2 was subjected to reduction with LiAlH4 in THF at 0 1Cfor 1 h to give the corresponding alcohol 2b, which upon furthercyclization by reaction with NaH in THF at 0 1C to room temperaturefor 4 h afforded oxazinanone 2c in 72% yield (over two steps).
The absolute configuration at Ca-position in 2 was assignedfrom the 1H NMR spectrum of 2c, wherein, the CbH protonresonated at d 3.72 with 3JCbH–C4H = 3.2 Hz, 3JCaH–CbH = 5.5 Hzand 3JCbH–NH6 = 1.4 Hz. Small values of 3JCaH–CbH and 3JCbH–C4H
are consistent with the dihedral angles Ha–Ca–Cb–Hb andH4–C4–Cb–Hb B 601, which provide emphatic support forCaH and CbH protons being stereochemically syn to each other.In addition, the nOe correlations Hb/H20a, Hb/H20b, H4/H20aand H4/H20b (Fig. 2) conclusively prove that the configurationat the Ca-stereocentre in b2,3-Caa 2 is ‘S’.
Conformational studies of 1 and 2
For monomer 1, 3JNH–CbH = 10.2 Hz, implies anti-periplanardisposition of the NH and CbH protons, which is consistent
with the dihedral angle C(O)–N–Cb–Ca (fb) B 1201 (Fig. 3A).Additionally small values of 3JCaH–CbH = 3.7 Hz correspond toN–Cb–Ca–C(O) (yb) B �601. The couplings 3JCaH–C30H are = 8.6 Hzand = 6.5 Hz, which along with one strong and other weakCbH/C30H nOe correlation, support predominance of dihedralangle Cb–Ca–C30–C20 (w10) B 1801 and allow prochiral assign-ment of C30H. Thus, the C30proton with larger 3JCaH–C30H andweak nOe correlation (W) with CbH was assigned as C30H( pro-S).
Scheme 1 Synthesis of monomer 2 and determination of the stereo-chemistry at C2.
Fig. 2 Structure of 2c as deduced from NMR data.
Fig. 3 Structures of amino acids: (A) 1 (C-allyl); (B) 2 (C-propargyl).
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
Similarly, the C30 proton with 3JCaH–C30H = 6.5 Hz and strong nOecorrelation (S) with CbH was assigned as a C30H( pro-R). Inaddition 3JCbH–C4H = 7.4 Hz, along with medium intensity nOecorrelation (M), NH/C1H, suggest structures with predominanceof N–Cb–C4–C3 (w1) B 601 (Fig. 3A).
For monomer 2, 3JNH–CbH = 9.9 Hz, justifies anti-periplanardisposition of the NH and CbH protons corresponding to afb B 1201 (Fig. 3B), while, 3JCaH–CbH = 3.8 Hz is consistent withyb B � 601. C30 protons display 3JCaH–C30H = 9.2 Hz and 5.7 Hz,which along with one strong (S) and other weak (W) nOecorrelation involving C30 protons (CbH/C30H, C30H/C3H) notonly enable us to make prochiral-assignments, but also fix thedihedral angle as w10 B 1801. Thus, the C30 proton with 3JCaH–
C30H = 9.2 Hz and showing weak nOe correlation C30H/CbH, wasassigned as C30H( pro-S). Similarly, the proton with 3JCaH–C30H =5.7 Hz and strong nOe correlation C30H/CbH was assigned asC30H( pro-R). In addition, 3JCbH–C4H = 6.8 Hz, along with mediumintensity (M) nOe correlation, NH/C1H, suggest structures withpredominance of N–Cb–C4–C3 (w1) B 601 (Fig. 3B). For 1 and 2,though it was not possible to derive the information on theconformation about Ca–C(O) bond, the constrained value ofother backbone dihedral angle appears to be an attractive optionto explore these monomers for obtaining robust foldamers.
Synthesis of peptides 4–17
The a/b2,3-peptides 4–17 were prepared from L-Ala and from theknown 117a and 2 by standard peptide coupling methods19 in
solution phase. Accordingly, esters 1 and 2 on exposure toCF3COOH in CH2Cl2 for 1 h, were converted into the corre-sponding amine salts 1a and 2a (Scheme 2). Coupling of acid 3,independently with amine salts 1a and 2a in the presence ofEDCI, HOBt and DIPEA in CH2Cl2 at 0 1C to room temperaturefor 6 h furnished the dipeptides 4 (80%) and 5 (74%), respec-tively. Base hydrolysis of peptides 4 and 5 independently, withLiOH afforded the acids 4a and 5a, while, 4 and 5 on treatmentwith CF3COOH in CH2Cl2 gave respective salts 4b and 5b. Acids4a and 5a independently on coupling (EDCI, HOBt and DIPEA)with the amine salt 3b (prepared from 3a on treatment withCF3COOH in CH2Cl2 at 0 1C for 2 h) afforded the tripeptides 6(74%) and 7 (78%) respectively (Scheme 2). Further, treatment ofpeptides 6 and 7 with CF3COOH in CH2Cl2 furnished the aminesalts 6a and 7a respectively. Similarly, coupling of acid 4a withthe amine salt 4b in the presence of HATU and DIPEA in CH2Cl2
at 0 1C to room temperature for 6 h furnished the tetrapeptide 8(69%), while, acid 5a on coupling (EDCI, HOBt and DIPEA) withsalt 5b gave the tetrapeptide 9 in 73% yield (Scheme 2).
To understand the impact of the side chain at C2 position inthe monomers 1 and 2, two tetrapeptides 10 and 11 were preparedfrom 1 and L-Ala. Accordingly, coupling (HATU and DIPEA) of theknown11c acid 18a with the salt 4b afforded the tetrapeptide 10(72%), while, acid 4a was coupled with the known11c salt 18b in thepresence of HATU and DIPEA to give tetrapeptide 11 (70%). Acid(CF3COOH) mediated reaction of 10 and 11 in CH2Cl2 furnishedthe respective amine salts 10a and 11a (Scheme 2).
Scheme 2 Synthesis of peptides 4–11.
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
In a further study, 12–17 were prepared to learn more aboutthe conformational behaviour of peptides. Accordingly, acid 5aon coupling (EDCI, HOBt and DIPEA) with amine salt 7a gavethe pentapeptide 12 (58%), while, a similar coupling of acid 4awith amine salt 6a did not result in the expected pentapeptide13 (Scheme 3). However, coupling of acid 18a with the salt 6aafforded the 13a (63%). Acid 4a on coupling reaction with theamine salts 11a and 10a in the presence of HATU and DIPEA inCH2Cl2 independently furnished the hexapeptides 14 (64%)and 16 (62%) respectively (Scheme 3).
Similarly, base (LiOH) hydrolysis of tetrapeptide 9 affordedthe acid 9a, on the other hand 9 on treatment with CF3COOH inCH2Cl2 was converted into amine salt 9b. Coupling of acid 9awith amine salt 18b in the presence of EDCI, HOBt and DIPEAin CH2Cl2 gave hexapeptide 15 (62%), while, coupling of acid18a with amine salt 9b afforded 17 in 67% yield (Scheme 3).
Conformational analysis
NMR studies of peptides 4–17 were carried out in B5 mMCDCl3 solution20 at 298 K, except 8 and 9, which were studied at288 K. 1H NMR spectra of most of the peptides show thepresence of another small isomer as was deduced fromexchange peaks in the ROESY spectra. However, due to thesmall population of the minor isomer (probably o2%), thedetailed studies were performed only on the major isomer.Peptides 4 and 5 have shown structural features similar to themonomers 1 and 2 respectively.20
Our earlier studies on variety of a/b-peptides,11c generating11/9-helices, displayed the formation of helix along the lengthof the oligomer with ‘a-b-a’ sequence at the C-terminus, while, a turnwas nucleated when the sequence was with ‘b-a-b’ at the C-terminus.14
Thus, the presentation has been organized by classifying the peptidesinto two families based on the above considerations.
In the following discussions, the numbering of the protonsin the peptides correspond to those shown in the Fig. 3(A) and(B). The figure and the text above also describe the dihedralangles, which have been defined explicitly at appropriate placein text. The numbering of the residues follow the usual con-vention of starting from the N-terminus.
Conformational studies on peptides 6/7 and 12/13a with ‘a-b-a’sequence at the C-terminus
Peptides 6 and 7 displayed wide dispersion of 1H NMR reso-nances indicating the presence of ordered secondary structure.It was additionally supported by significant populations of thepeptides having NH(2) and NH(3) participating in H-bonding,as was deduced from of their chemical shifts (dNH) andvariation (DdNH) from solvent titration studies21 (performedby adding sequentially up to 33% (v/v) DMSO-d6 to CDCl3
solution). For both the peptides 3JNH–CbH 4 10 Hz are consis-tent with the dihedral angle C(O)–N–Cb–Ca (fb) B 1201.21
Further, a strong CaH(2)/NH(3) nOe correlation is supportiveof a Cb–Ca–C(O)–NH (cb) B�1201. Small values of 3JCaH–CbH B2 Hz along with nOe correlation, NH(2)/NH(3) (M), and theinformation on dihedral angles deduced above providesemphatic support for N–Cb–Ca–C(O) (yb) B 601. A transientright-handed 11/9-mixed helix, consistent with the above data,was further supported by the nOe correlations, CaH(1)/NH(3)(M), CaH(1)/NH(2) (S). Preponderance of N–Cb–C4–C3 (w1) B1801 was deduced from 3JCbH–C4H B 10 Hz, which differ fromthose for the monomers 1 and 2.
For the pentapeptides 12 and 13a, except for the firstresidue, all other amide protons have propensity to H-bond,since they displayed dNH 4 7 ppm and small change in theirchemical shifts (DdNH) (DdNH o 0.38 ppm and 0.69 ppmfor 12 and 13a respectively).20,21 For the b3-Caa residues,3JNH–CbH 4 9.2 Hz and 3JCaH–CbH o 2.9 Hz along with strongCaH(2)/NH(3), CaH(3)/NH(4) and CaH(4)/NH(5) nOe correla-tions as well as medium range nOes NH(2)/NH(3) and NH(4)/NH(5) are consistent with fb B1201, cb B� 1201 and yb B 601,justifying an 11/9-helix. The 3JNH–CaH for the L-Ala residue inthe middle is 6.1 and 5.4 Hz for 12 and 13a respectively,implying agreement with high propensity of C(O)–N–Ca–C(O)(fa) B�701, expected for an 11/9-helix. The preponderance of aright-handed 11/9-mixed helix, with an 11/9/11/9-H-bondedpattern was further supported emphatically, by the characteristicnOes, NH(2)/NH(3), NH(4)/NH(5), CaH(1)/NH(3) and CaH(3)/NH(5). Thus, both the peptides 12 and 13a show the presenceof a right-handed 11/9-helix.
Scheme 3 Synthesis of peptides 12–17.
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
Restrained molecular dynamics (MD) calculations were carriedout using the distance restraints derived qualitatively from theROESY data and the dihedral angle constraints deduced fromcouplings.20 In view of the findings from the nOe correlations andthe couplings in the NMR spectra, the calculations were initiatedwith model structures having right-handed 11/9-helical folds.Stereoview of the superposition of a set of 10 best structures for12 are presented in Fig. 4, having backbone and heavy atom RMSDof 0.86� 0.81 and 0.86� 0.60 Å. The corresponding values for 13aare 0.47 � 0.27 and 0.99 � 0.36 Å. The helices appear to be lessrobust compared to those obtained with a/b-peptides derivedfrom b3-amino acids. The 9-atom H-bonded pseudo ringsNH(2)� � �CO(3), appear to be larger than prescribed for theH-bonds (2.5 Å). On the other hand, the MD calculations appearto suggest presence of NH(4)� � �O(CH3) H-bonds, instead of theexpected NH(4)� � �CO(5) H-bond. This may be a reflection/artefactof inadequate number of constraints involving the residues in thetermini as well as the fraying.
In 12, 3JCaH–C30H are 10.0 and 5.9 Hz for b2,3-Caa(2) and 10.8 and4.8 Hz for b2,3-Caa(4), which permit to make prochiral-assignmentsof C30H protons and fix w10 B 1801. Likewise, 3JCbH–C4H 4 9.3 Hzfor b2,3-Caa residues imply preponderance of w1 B 1801. For 13a,with allyl side chain at b2,3-Caa(2), w1 was deduced as B1801 from3JCbH–C4H = 10.3 Hz, while due to spectral overlaps like in 6, noinformation could be obtained on w10. For b3-Caa(4), 3JCbH–C4H =10.2 Hz, justify preponderance of w1 B 1801.
Conformational analysis of peptides 8/9, 10/11 and 14–17 with‘b-a-b’ sequence at the C-terminus
In peptides 8 and 9, the amide protons in the middle displayeddNH 4 7 ppm. The values of DdNH for NH(2) and NH(3) werevery similar to those in the tripeptides 6 and 7, thus confirmingthe predominance of the structure, involving these amideprotons in H-bonding. 3JNH–CbH 4 9.8 Hz and strong CaH(2)/NH(3) nOe correlations justify fb B 1201 and cb B �1201.Small values of 3JCaH–CbH (o2.3 Hz) along with the presence of
NH(2)/NH(3) correlation support a single conformer aroundCaH–CbH with y B 601. 3JNH–CaH B 7 Hz (6.6 to 7.3 Hz) for theL-Ala residues again suggests a probable conformational aver-aging. However, in view of the nucleation of a right-handed11/9-helix in 6 and 7, which ideally has fa B �701, thesecouplings are consistent with the propensity of such structures.Thus, the information on H-bonding and dihedral angles,along with strong sequential nOes, CaH(1)/NH(2), CaH(2)/NH(3) and CaH(3)/NH(4) and medium range nOe correlations,NH(2)/NH(3) and NH(3)/CaH(1), with medium intensity yetagain support the preponderance of structures with right-handed 11/9-mixed helical folds in 8 and 9. Though the pep-tides had a ‘b-a-b’ sequence at the C-terminus, no signature of aturn was observed.
For 8, the information on w10 in b2,3-Caa(2) could not beobtained, while, for b2,3-Caa(4), preponderance of w10 B 1801was deduced. Distinctly different conformational behaviourwith 3JCbH–C4H = 9.6 and 6.2 Hz for b2,3-Caa(2) and b2,3-Caa(4)respectively was noticed, implying the preponderance of w1 B1801 for the former and B 601 for the latter. Similar conforma-tional space was traversed by w1 for the b2,3-Caa residues in 9 aswell. However, unlike 8, definitive information on w10 B 1801could be deduced for 9, with 3JCaH–C30H having large and smallvalues (9.9 and 5.1 Hz).
To understand the impact of side chain on the secondarystructures, peptides 10 and 11, having b3-Caa residue at secondand fourth residues respectively (thus having no substitution atCa), were studied. For 10, amide protons in the core of the peptideagain displayed dNH 4 7 ppm and significantly small DdNH o0.40 ppm,22 implying their involvement in H-bonding. StrongCaH(2)/NH(3) nOe correlation along with 3JNH–CbH 4 9.0 Hz areconsistent with fb B 1201 and cb B �1201. In addition, nOecorrelation NH(2)/NH(3) and 3JCaH–CbH o 5.3 Hz justify prepon-derance of ybB 601. Like 8 and 9, 3JNH–CaH = 6.4 and 7.1 Hz for theL-Ala residues support preponderance of structures with fa B�701, which along with above structural information and
Fig. 4 Stereoview of superimposed structures for peptide 12 (for the clarity, the protons are removed).
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
characteristic nOes, NH(2)/NH(3), CaH(1)/NH(3) further support aputative 11/9-mixed helix, involving the first three residues. Inter-estingly, very weak medium range nOe correlations like: CaH(1)/NH(4), C4H(2)/NH(4) and NH(3)/NH(4), involving the last residuewere noticed. A careful analysis of the data revealed that despitefraying at the termini, a turn is being induced, by a 11/9-helix at theN-terminus, resulting in a transient ‘helix-turn’ motif in peptide 10.For the peptide 11, the results are qualitatively similar to those for10. Very weak signatures of the helix-turn are distinctly noticed(ESI†). MD calculations for 8–11, could not be carried out in view ofthe poorer definition of the secondary structures.
As discussed, the signatures of the turn were conspicuous bytheir absence in 8 and 9 with both the b-residues having bulkysubstitution at CaH and CbH, while, in 10 and 11, with only oneb2,3-residue, the nOe data indicated the presence of a putativeturn. It appears that the steric crowding by the substituentsinfluences the stability of the regular structures.
For peptide 10, large and small 3JCbH–C30H values for b2,3-Caa(4)allow us to fix the w10 B 1801. Like 8, 3JCbH–C4H = 9.4 Hz forb3-Caa(2) and 6.2 Hz for b2,3-Caa(4) are consistent with prepon-derance of w1 B 1801 and B601 respectively. On the other handfor peptide 11, due to spectral overlap, information on w10 was notavailable, whereas, from 3JCbH–C4H, w1 B 1801 and B601 werededuced for b2,3-Caa(2) and b3-Caa(4) 601 respectively.
In view of the tetrapeptides, discussed above, displayingrather weak turn structures, larger hexapeptides, 14–17 withvaried substitution patterns, were studied. Peptides 14 and 15,with substituent pattern similar to those of 8 and 9, with b2,3-Caas as the second and fourth residues were first investigated.These peptides had several amides with dNH 4 7 ppm, thoughsmall value of DdNH o 0.08 ppm were found only for NH(2)and NH(4). NH(3) and NH(5) displayed DdNH Z 0.99 ppm,reflecting their weak H-bonding character. 3JNH–CbH involvingthe NH(2), NH(4) and NH(6) are respectively 10.0 (10.2), 10.1(9.7) and 8.2 Hz (8.2 Hz) for 14 (15). Thus, though for b2,3-Caa(2)
and b2,3-Caa(4) residues the backbone dihedral angles areconstrained with f B 1201, for the b2,3-Caa(6) residue, thereappears to be likely deviation in f or a reflection of possibleaveraging around Ca–Cb. In agreement with these conclusions,the 3JCaH–CbH for b2,3-Caa(2) and b2,3-Caa(4) are B2 Hz, pointing toconstrained values of y B 601, while for b2,3-Caa(6), a possibleconformational averaging is reflected in values of 5.2–6.3 Hz. Forthe L-Ala residues, 3JNH–CaH = 5.8–7.3 Hz might reflect conforma-tional averaging, yet, they are consistent with the predominanceof a fa B �701, expected for the 11/9-helix. The characteristicnOes, NH(2)/NH(3), NH(4)/NH(5), CaH(1)/NH(3) and CaH(3)/NH(5) with significant intensities point to the presence of popula-tions of 11/9-mixed helical folds. Like 8 and 9, the signatures ofa turn at the C-terminus were conspicuous by their absence. Itshould however be mentioned that the spectral overlaps oftencloud observation of distinct nOe correlations.
For 14, the MD calculations showed 11/9/11/9-H-bondedstructures, with 11- and 9-atom H-bond lengths of B2.2 andB2.60 Å respectively. The 10 best structures (Fig. 5) for 14 had abackbone and heavy atom RMSD of 0.32 � 0.39 and 0.60 � 0.46 Årespectively, while, for 15, the values of backbone and heavy atomRMSD 0.54 � 0.28 and 1.24 � 0.60 Å respectively, point towards aless robust helical structure. These structures also displayedrather large H-bond lengths.
For 14, 3JCbH–C4H B 9.5 Hz for both the b2,3-Caa(2) and b2,3-Caa(4) imply w1 = 1801, while, due to the spectral overlapinformation on w10 could not be obtained. Also, 3JCbH–C4H =6.5 Hz for b3-Caa(6) implies preponderance of w1 B 601. For 15,large and small values of 3JCbH–C30H for the both b2,3-Caa(2) andb2,3-Caa(4) allow us to fix w10 B 1801.
For 16, the second and sixth residues are b2,3-amino acid(an allyl functionality at C2) and the fourth residue is a b3-Caa.The residues in the middle have dNH 4 7 ppm and DdNHo 0.32 ppm,21 supporting their participation in H-bonding.The large values of 3JNH–CbH (49.3 Hz) are consistent withfbB 1201.
Fig. 5 Stereoview of superimposed structures for peptide 14 (for the clarity, the protons are removed).
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
Strong CaH(2)/NH(3) and CaH(4)/NH(5) nOes again satisfycb B 1201. Small values of 3JCaH–CbH [1.6 and 3.9 Hz for b2,3-Caa(2) and b2,3-Caa(6) residues respectively and 2.1 and 5.2 Hz forb3-Caa(4) residue] along with the presence of nOe correlationsNH(2)/NH(3) and NH(4)/NH(5) support a single conformer aroundCaH–CbH with yb B 601. A probable conformational averaginginvolving L-Ala residues was indicated with 3JNH–CaH between6.3–7.2 Hz for the L-Ala residues, though like other smallerpeptides, suggestion of the propensity of an 11/9-helix with fa
B �701 is also implied. Above deductions along with character-istic nOes, NH(2)/NH(3), NH(4)/NH(5), CaH(1)/NH(3) and CaH(3)/NH(5), provide additional support for the preponderance of11/9-helix. As in tetrapeptides 10 and 11, feeble signatures of aturn at the C-terminus were observed in the form of weak CaH(3)/NH(6), C4H(4)/NH(6), NH(4)/NH(6) and NH(5)/NH(6) nOes.
The MD calculations on peptide 16 showed signatures of ahelix-turn structure (Fig. 6A). The 10 best structures had abackbone and heavy atom RMSD of 0.77 � 0.66 and 0.87 �0.57 Å respectively. The 11-atom H-bond lengths were B2.5 Å,while, the 9-atom H-bond length was B2.8 Å, showing lessrobust nature of the structure. The average H-bond length forNH(4)� � �CO(6) H-bond in the 13-mr turn was B2.5 Å (Fig. 6B).
For 16, due to the spectral overlap for b2,3-Caa(2), information onw10 could not be obtained. For b2,3-Caa(6) the large and small3JCbH–C30H values allow to fix w10 B 1801. Like 14, 3JCbH–C4H 410.1 Hz for b2,3-Caa(2) and b3-Caa(4) imply w1 B 1801 and3JCbH–C4H = 6.4 Hz for b2,3-Caa(6) justifies w1 B 601.
Peptide 17 has two b2,3-Caa residues with bulky side chainsin closer proximity in the turn region. The amide protons in themiddle have dNH 4 7 ppm, while only three of them haveDdNH o 0.60 ppm [NH(2), NH(3) and NH(4)], consistent withtheir participation in H-bonding. For b-residues, large values of3JNH–CbH (49.6 Hz) support fb B 1201, while strong nOecorrelations, CaH(2)/NH(3) and CaH(4)/NH(5) imply cb B �1201.Small values of 3JCaH–CbH [1.9 and 3.6 Hz for b2,3-Caa(4) and b2,3-Caa(6) residues respectively and 3.7 and 4.7 Hz for b3-Caa(2)residue] along with the presence of nOes, NH(2)/NH(3) andNH(4)/NH(5), justify y B 601. Though for the Ala(3), the 3JNH–CaH
5.1 Hz is consistent with values of fa B �701, expected for an11/9-helix, the 3JNH–CaH B 7.0 Hz for other L-Ala residues reflects
probable conformational averaging. However, the preponderanceof a mixed 11/9-helix is further supported by the characteristicnOes, NH(2)/NH(3), NH(4)/NH(5), CaH(1)/NH(3) and CaH(3)/NH(5).In addition, except for the presence of NH(5)/NH(6), the othersignatures of a turn at the C-terminus, like those in 16, were notdistinct, due to spectral overlap. Comparing the data for 16 and 17,it appears that the 11/9-helix in the former is more robust.
For 17, the large and small values of 3JCbH–C30H for b2,3-Caa(4)and b2,3-Caa(6) support the w10 B 1801. Further, the 3JCbH–C4H
4 9.7 Hz for both the b3-Caa(2) and b2,3-Caa(4) impliesw1 B 1801, while, 3JCbH–C4H = 6.4 Hz for b2,3-Caa(6) implies w1B 601. Interestingly, for all the hexapeptides irrespective ofthe substitution pattern, w1 B 1801 for the second andfourth residues, while, for the sixth residue preponderance ofw1 B 601 was noticed.
Thus, a comparison of the hexapeptides 14–17 suggestedthat a well defined structure was obtained when the two bulkyb2,3-Caa residues are far apart, as was the case in 16, thushaving little steric interaction. The more robust structure in 16also displays very distinctly the features of a turn at theC-terminus unlike the other hexapeptides, where, the confor-mational averaging clouds the signatures of weak structuralfeatures. On the other hand, the pentapeptides 12 and 13a,clearly display an 11/9-helix.
An interesting observation emerged from the studies, thatthe stereospecific assignments of the C30 protons in peptideshaving the allyl groups in b2,3-Caa(2) residues (peptides 6, 8, 11,13a, 14, and 16) could not be achieved, as 3JCaH–C30H couplingsand characteristic nOe correlations (CbH/C30H, C30H/C3H)were not distinctive. However, in these residues with3JCbH–C4H 4 9 Hz, predominance of N–Cb–C4–C3 (w1) B 1801is implied.20 On the other hand, for peptides with allyl groupsin b2,3-Caa(4) and b2,3-Caa(6) (peptides 8, 10, 13a, 14 and 16)and those containing propargyl groups (except 7), it was possibleto make stereospecific assignments of C30 protons. In addition,for tetra- and hexapeptides, the terminal b-residues irrespectiveof whether it is a b2,3-Caa (with allyl or propargyl) or a b3-Caaresidue, with 3JCbH–C4H o 6.5 Hz, imply preponderance ofw1 B 601, which differs from value of B1801 observed for theother b-residues, including those in the tri- and pentapeptides.
Fig. 6 (A) Stereoview of superimposed structures for peptide 16 (for the clarity, the protons are removed); (B) details of one of the structures depictingNH(4)� � �CO(6) (H-bond with dotted line) in the ‘b-a-b’ turn.
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
In all the peptides, the sugar pucker of b3-Caa and b2,3-Caaresidues appears to be similar. The 3JC1H–C2H B 3 Hz, 3JC2H–C3H
B 0 Hz and 3JC3H–C4H B 4 Hz and strong nOe correlations,Me(pro-R)/C1H, Me(pro-R)/C2H and weak Me(pro-S)/C4H areconsistent with a 3T2 pucker of the sugar ring and an envelopeconformation of the isopropylidene ring, similar to our earlierfindings.14c
Conclusions
In conclusion, a new class of b2,3-Caas with Ca-allyl andpropargyl groups were prepared and utilized in the synthesisof hybrid 1 : 1 a/b2,3-peptides with L-Ala. The tri- and penta-peptides with ‘a-b-a’ sequence at the C-terminus have shownthe presence of a right-handed 11/9-helix. On other hand,the tetra- and hexapeptides with the ‘b-a-b’ sequence at theC-terminus revealed the presence of weak turn. Despite the factthat the b2,3-substitutions impart strong backbone constraintsabout Ca–Cb, the presence of weak helical and turn structures inthe new class of a/b2,3-peptides is a result of the steric inter-actions arising due to C-allylic/propargylic side chains. Such b2,3-disubstituted amino acids are likely to find diverse use in thefurther application of these monomers in the peptide design.
ExperimentalGeneral experimental
NMR spectra (1D and 2D experiments) for peptides 4–17 wereobtained at 600 MHz (1H), and at 150 MHz (13C). Chemicalshifts are reported in d scale with respect to internal TMSreference. Information on hydrogen bonding in CDCl3 wasobtained from solvent titration studies at 298 K by sequentiallyadding up to 300 mL of DMSO-d6 in 600 mL of CDCl3 solution ofpeptides. States-TPPI procedure22 was used to run various NMRexperiments in the phase sensitive mode using standard pro-grams in the library provided by the instrument manufacturer.The ROESY experiments were performed with mixing times of300 ms using a continuous spin-locking field of about 2.5 kHz. TheTOCSY experiments were performed with the spin locking field ofabout 10 kHz and a mixing time of 80 ms. The 2D data wereprocessed with Gaussian apodization in both the dimensions.
The Insight-II (97.0)/Discover program23 was used for con-struction of the molecular model and for structural analysis ofdifferent obtained conformations, including molecular model-ling calculations and energy minimization. The CVFF forcefield with default parameters was used throughout the calcula-tions with the aid of a distance-dependent dielectric constantwith dielectric constant (e) = 4.8 for CDCl3.
For restrained molecular dynamics (MD) calculation, thedistance as well as dihedral angle constraints were used. Due tospectral overlap in the ROESY spectrum, the nOe intensitiescould not be obtained accurately. Thus, the distance con-straints were deduced from the cross peak in the ROESY spectraqualitatively. The dihedral angle constraints were deducedfrom the couplings as well as nOes cross peaks from the ROESY
spectrum.21 In view of the support for the 11/9-helices from theNMR data for these peptides, the molecular model was builtbased on the backbone dihedral angles from the theoreticalstudies by Hofmann et al.11d for the initiation of the dynamics.Initial minimizations of the structures were carried out withsteepest descent, followed by conjugate gradient methods fora maximum of 1000 iterations each or RMS deviation of0.001 kcal mol�1, whichever was earlier. The energy-minimizedstructures were then subjected to MD simulations. A number ofinter-atomic distance and torsional angle constraints obtainedfrom NMR data were used as restraints in the minimization aswell as MD runs. For MD runs, a temperature of 300 K was used.The molecules were initially equilibrated for 50 ps and subse-quently subjected to a 1 ns dynamics with a step size of 1 fs,sampling the trajectory at equal intervals of 10 ps to generate106 structures. These structures were further subjected to energyminimization using the above-mentioned protocol and ten of thebest possible structures are superimposed for display.
Synthesis of monomer 2 and its derivative 2ctert-Butyl (1S,2S)-2-(methoxycarbonyl)-1-((3aR,5R,6S,6aR)-tetrahydro-
6-methoxy-2,2-dimethylfuro[2,3-d][1,3]dioxol-5-yl)pent-4ynylcarbamate(2). To a solution of N,N-diisopropyl amine (0.63 mL, 4.66 mmol) inTHF (5 mL) at�78 1C, n-BuLi (2.6 M solution in n-hexane, 1.79 mL,4.66 mmol) was added and stirred at 0 1C for 30 min. A solution of2a (0.5 g, 1.33 mmol) in THF (5 mL) was added at �78 1C, and itwas allowed to stir at the same temperature for 30 min. Propargylbromide (0.29 mL, 1.99 mmol) was added to the reaction mixture at�78 1C and stirred the reaction mixture for an additional 2 h. It wasquenched with cold aq. NH4Cl (8 mL) and extracted with EtOAc(2 � 20 mL). The extracts were washed with water (2 � 10 mL),brine (10 mL) and dried (Na2SO4). Solvent was evaporated underreduced pressure and purified residue by column chromatography(60–120 mesh silica gel, 12% ethyl acetate in pet. ether) to give 2(0.24 g, 42%) as a light yellow syrup; [a]20
D =�60.49 (c 0.3, CHCl3); IR(neat): 3287, 2981, 2935, 1719, 1505, 1442, 1372, 1225, 1166, 1118,1081, 1019, 857 cm�1; 1H NMR (300 MHz, CDCl3): d 5.89 (d, 1H,J = 3.6 Hz, C1H), 5.05 (d, 1H, J = 9.4 Hz, NH), 4.56 (s, 1H, C2H), 4.38–4.31 (m, 1H, CbH), 4.08–4.03 (m, 1H, C4H), 3.73 (s, 3H, COOMe),3.68 (d, 1H, J = 3.1 Hz, C3H), 3.37 (s, 3H, OMe), 2.83–2.79 (m, 1H,CaH), 2.69–2.59 (m, 1H, CH2), 2.53–2.43 (m, 1H, CH2), 1.99 (t, 1H,J = 2.2 Hz, acetylinic), 1.46 (s, 12H, Boc, CH3), 1.31 (s, 3H, CH3); 13CNMR (75 MHz, CDCl3, 298 K): d 172.9, 155.7, 112.1, 111.5, 104.7,84.3, 81.2, 79.6, 69.9, 57.6, 52.0, 50.0, 47.0, 29.7, 28.3, 26.7, 26.2,18.8; HRMS (ESI): m/z calculated for C20H32NO8 (M + Na) 436.1947,found 436.1950.
(4S,5S)-4-((3aR,5R,6S,6aR)-6-Methoxy-2,2-dimethyl-tetrahy-drofuro-[2,3d][1,3]dioxol-5-yl)-5-(prop-2-ynyl)-1,3-oxazinan-2-one (2c). To astirred suspension of LiAlH4 (0.33 g, 2.15 mmol) in THF (7 mL)at 0 1C, a solution of 2 (0.48 g, 1.15 mmol) in THF (8 mL) was addeddropwise under a nitrogen atmosphere and stirred at room tem-perature for 1 h. The reaction mixture was cooled to 0 1C, treatedwith saturated aq. Na2SO4 solution (10 mL) and filtered. It waswashed with EtOAc (50 mL) and the filtrate was dried (Na2SO4).Solvent was evaporated to give the corresponding alcohol 2b, whichwas directly used for the next reaction.
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
To an ice cooled (0 1C) suspension of NaH (42 mg,1.75 mmol, 60% w/w dispersion in paraffin oil) in THF(2 mL), a solution of 2b (0.45 g, 1.16 mmol) in THF (3 mL)was added drop wise and stirred at room temperature for 4 h.The reaction mixture was quenched with saturated aq. NH4Cl(4 mL) and extracted with ethyl acetate (2 � 15 mL). Thecombined organic layers were washed with water (10 mL), brine(10 mL) and dried (Na2SO4). Solvent was evaporated andpurified the residue by column chromatography (60–120 meshsilica gel, 50% ethyl acetate in pet. ether) to afford 2c (0.26 g,72%) as a light yellow syrup; [a]20
D = �42.94 (c 0.3, CHCl3); IR(neat): 3287, 2981, 2935, 1719, 1505, 1442, 1372, 1225, 1166,1118, 1081, 1019, 857 cm�1; 1H NMR (300 MHz, CDCl3): d 5.93(d, 1H, J = 3.8 Hz, C1H), 5.82 (brs, 1H, NH), 4.59 (d, 1H, J = 3.8 Hz,C2H), 4.42 (dd, 1H, J = 3.4, 11.1, 14.6 Hz, C4H), 4.18–4.15 (m, 2H,OCH2), 3.82 (d, 1H, J = 3.8 Hz, CbH), 3.72–3.71 (m, 1H, C3H), 3.44(s, 3H, OMe), 2.48–2.37 (m, 2H, CH2), 2.23–2.17 (m, 1H, CaH), 2.08(t, 1H, J = 2.6 Hz, acetylenic), 1.48 (s, 3H, CH3), 1.33 (s, 3H, CH3);13C NMR (75 MHz, CDCl3, 298 K): d 153.8, 111.9, 104.8, 86.1, 81.0,79.7, 79.6, 71.3, 67.5, 57.5, 53.2, 33.2, 26.8, 26.1, 19.1; HRMS (ESI):m/z calculated for C15H22NO6 (M + H) 312.1447, found 312.1440.
Synthesis of peptidesBoc-L-Ala-b2,3-Caa-OMe (4). A solution of 3 (0.33 g, 1.73 mmol),
HOBt (0.30 g, 2.16 mmol) and EDCI (0.41 g, 2.16 mmol) in dryCH2Cl2 (8 mL) was stirred at 0 1C for 15 min, sequentially treatedwith salt 1a [prepared from 1 (0.60 g, 1.44 mmol) on treatmentwith CF3COOH (0.6 mL) in CH2Cl2 (4 mL) at 0 1C] and DIPEA(0.50 mL, 2.89 mmol) and stirred for 6 h. The reaction mixturewas quenched with satd. aq. NH4Cl (15 mL) at 0 1C and dilutedwith CHCl3 (25 mL). It was sequentially washed with 1 N HCl(15 mL), water (15 mL) and aq. NaCl solution (15 mL). Theorganic layer was dried (Na2SO4), evaporated and purified theresidue by column chromatography (60–120 mesh silica gel, 50%ethyl acetate in pet. ether) to afford 4 (0.56 g, 80%) as a lightyellow syrup; [a]20
D = �27.4 (c 0.37, CHCl3); IR (neat): 3360, 2921,2851, 1719, 1515, 1460, 1370, 1246, 1167, 1079, 1022, 856,772 cm�1; 1H NMR (500 MHz, CDCl3, 298 K): d 6.61 (d, 1H, J =9.8 Hz, NH), 5.86 (d, 1H, J = 3.7 Hz, C1H), 5.82–5.68 (m, 1H,olefinic), 5.15-5.02 (m, 2H, olefinic), 5.05 (d, 1H, J = 9.8 Hz,NHBoc), 4.63–4.55 (m, 1H, CH), 4.54 (d, 1H, J = 3.7 Hz, C2H),4.15–4.09 (m, 2H, CbH and C4H), 3.70 (s, 3H, OCH3), 3.64 (d, 1H,J = 3.4 Hz, C3H), 3.35 (s, 3H, OCH3), 2.68–2.62 (m, 1H, CaH),2.46–2.22 (m, 2H, allylic CH2), 1.46 (s, 3H, CH3), 1.44 (s, 9H, Boc),1.33 (d, 3H, J = 6.8 Hz, CH3), 1.30 (s, 3H, CH3); 13C NMR(150 MHz, CDCl3, 298 K): d 174.1, 172.5, 155.2, 134.6, 117.3,111.5, 84.2, 81.1, 79.7, 64.5, 57.4, 51.7, 50.3, 48.0, 46.5, 45.5, 33.4,28.3 (3C), 26.7, 26.2, 18.5; HRMS (ESI+): m/z (M+ + Na) calculatedfor C23H38N2O9 509.24695, found 509.24712.
Boc-L-Ala-(S)-b2,3-Caa-OMe (5). A solution of 2 (0.48 g,1.15 mmol) and CF3COOH (0.5 mL) in CH2Cl2 (2 mL) was stirredat 0 1C to room temperature for 2 h. Solvent was evaporatedunder reduced pressure and resulting salt 2a was dried underhigh vacuum and used as such for further reaction.
A solution of 3 (0.22 g, 1.15 mmol), HOBt (0.18 g, 1.38 mmol)and EDCI (0.26 g, 1.38 mmol) in dry CH2Cl2 (6 mL) was stirred
at 0 1C for 15 min, sequentially treated with salt 2a [preparedfrom 2 (0.48 g, 1.15 mmol) on treatment with CF3COOH(0.5 mL) in CH2Cl2 (2 mL) at 0 1C] and DIPEA (0.20 mL,1.10 mmol) and stirred for 6 h. Work up as described for 4and purification of the residue by column chromatography(60–120 mesh silica gel, 40% ethyl acetate in pet. ether) afforded5 (0.41 g, 74%) as a brown syrup; [a]20
D = �72.82 (c 0.5, CHCl3); IR(KBr): n 3308, 2983, 2936, 1720, 1518, 1454, 1373, 1249, 1167,1120, 1080, 1023, 857, 756, 639, 523 cm�1; 1H NMR (CDCl3,500 MHz): d. 6.55 (d, 1H, J = 9.4 Hz, NH-2), 5.87 (d, 1H, J = 3.7 Hz,C1H-1), 5.14 (s, 1H, NH-1) 4.69–4.63 (m, 1H, CbH), 4.55 (d, 1H,J = 3.7 Hz, C2H), 4.17–4.08 (m, 2H, CaH-1, C4H), 3.74 (s, 3H,COOMe), 3.67 (d, 1H, J = 3.3 Hz, C3H), 3.35 (s, 3H, OMe), 2.25(m, 1H, CaH-2), 2.64–2.54 (m, 1H, CH2) 2.49–2.40 (m, 1H, CH2),2.00 (t, 1H, J = 2.2 Hz, acetylinic) 1.46 (s, 3H, Me), 1.43 (s, 9H, Boc),1.34 (s, 3H, Me), 1.32 (s, 1H, CH3), 1.30 (d, 3H, J = 4.1 Hz, Me), 1.25(t, 2H, J = 7.9 Hz, CH3); 13C NMR (CDCl3, 150 MHz): d 172.8, 172.6,155.3, 111.6, 104.7, 84.3, 81.0, 79.3, 70.1, 57.6, 52.2, 50.2, 48.1, 46.5,29.6, 28.3(3C), 26.7, 26.2, 18.9, 18.5; HRMS (ESI+): m/z calculated forC23H37N2O9 (M+ + H) 485.2499, found 485.2484.
Boc-L-Ala-b2,3-Caa-L-Ala-OMe (6). A cooled (0 1C) solution of 4(0.38 g, 0.78 mmol) in THF : MeOH : H2O (3 : 1 : 1) (5 mL) wastreated with LiOH (46 mg, 1.95 mmol) and stirred at roomtemperature. After 1 h, pH was adjusted to 2–3 with 1 N HClsolution at 0 1C, and the solution was extracted with ethylacetate (2 � 40 mL). The organic layer was dried (Na2SO4) andevaporated to give 4a (0.35 g, 96%) as a colourless solid.
A solution of acid 4a (0.15 g, 0.31 mmol), HOBt (0.06 g,0.47 mmol) and EDCI (0.09 g, 0.47 mmol) in dry CH2Cl2 (5 mL)was stirred at 0 1C for 15 min and treated with the amine salt 3band DIPEA (0.14 mL, 0.79 mmol) under nitrogen atmospherefor 6 h. Work up as described for 4 and purification of theresidue by column chromatography (60–120 mesh silica gel,80% ethyl acetate in pet. ether) afforded 6 (0.13 g, 74%) as awhite solid; m.p. 99–101 1C; [a]20
D = +35.6 (c 0.13, CHCl3); IR(KBr): 3338, 3302, 3055, 2986, 2939, 1726, 1676, 1544, 1459,1373, 1325, 1233, 1168, 1078, 1016, 911, 859, 617 cm�1; 1H NMR(600 MHz, CDCl3, 298 K): d 7.51 (d, 1H, J = 10.5 Hz, NH-2), 7.46(d, 1H, J = 8.1 Hz, NH-3), 5.87 (d, 1H, J = 3.9 Hz, C1H), 5.79 (tdd,1H, J = 6.8, 10.4, 17.2, olefinic), 5.10 (qd, 1H, J = 1.5, 17.2,olefinic), 5.02 (qd, 1H, J = 1.5, 10.4, olefinic), 5.01 (d, 1H,J = 7.0 Hz, NHBoc), 4.64 (dq, 1H, J = 7.6, 8.1 Hz, CaH-3), 4.58(d, 1H, J = 3.9 Hz, C2H), 4.54 (td, 1H, J = 2.1, 10.5 Hz, CbH), 4.09(dd, 1H, J = 3.2, 10.5 Hz, C4H-2), 3.95 (d, 1H, J = 3.2 Hz, C3H), 3.87(qt, 1H, J = 7.0 Hz, CaH-1), 3.73 (s, 3H, OMe), 3.40 (s, 3H, OMe),2.48 (td, 1H, J = 2.1, 8.2 Hz, CaH-2), 2.39 (m, 2H, allylic CH2), 1.50(d, 3H, J = 7.5 Hz, CH3-3), 1.45 (s, 3H, CH3), 1.40 (s, 9H, Boc), 1.36(d, 3H, J = 7.0 Hz, CH3-1), 1.30 (s, 3H, CH3); 13C NMR (150 MHz,CDCl3, 298 K): d 175.7, 172.9, 172.8, 155.9, 135.0, 117.0, 111.5,104.8, 83.5, 81.5, 80.2, 80.1, 57.3, 52.6, 51.5, 49.9, 48.9, 48.6, 32.7,28.3 (3C), 26.8, 26.5, 17.0, 16.2; HRMS (ESI+): m/z (M+ + Na)calculated for C26H43N3O10 580.28407, found 580.28406.
Boc-L-Ala-(S)-b2,3-Caa-L-Ala-OMe (7). A solution of ester 5(0.2 g, 0.41 mmol) in THF : MeOH : H2O (3 : 1 : 1) (4 mL) was
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
treated with LiOH (24 mg, 1.03 mmol) and stirred at roomtemperature. Work up as described for 6 gave 5a (0.18 g, 96%)as a colourless solid, which was used for the next reactionwithout further purification.
A solution of 5a (0.05 g, 0.38 mmol), HOBt (0.06 g, 0.45 mmol)and EDCI (0.09 g, 0.45 mmol) in dry CH2Cl2 (4 mL) was stirred at0 1C for 15 min and treated with salt 3b and DIPEA (0.09 mL,0.57 mmol) under nitrogen atmosphere for 5 h. Work up asdescribed for 4 and purification of the residue by columnchromatography (60–120 mesh silica gel, 80% ethyl acetate inpet. ether) afforded 7 (0.17 g, 78%) as a white solid; m.p. 102–109 1C; [a]20
D = +51.32 (c 0.1 in CHCl3); IR (KBr): n 3360, 3231, 2983,2931, 1733, 1660, 1534, 1457, 1376, 1325, 1231, 1165, 1080, 1023,857, 756, 639, 523 cm�1; 1H NMR (CDCl3, 500 MHz): d 7.43 (d, 1H,J = 8.0 Hz, NH-3), 7.36 (d, 1H, J = 10.2 Hz, NH-2), 5.79 (d, 1H,J = 3.7 Hz, C1H-2), 4.97 (d, 1H, J = 6.5 Hz, NH-1), 4.63 (dd, 1H,J = 7.3, 1.5 Hz, CaH-3), 4.53 (d, 1H, J = 3.6 Hz, C2H), 4.46–4.41(m, 1H, CbH), 3.98 (dd, 1H, J = 9.8, 3.2 Hz, C4H), 3.93 (d, 1H,J = 3.2 Hz, C3H), 3.82 (dd, 1H, J = 13.9, 6.5 Hz, CaH-1), 3.75 (s, 3H,COOMe), 3.45 (s, 3H, OMe), 2.66–2.61 (m, 1H, CaH-2), 2.43–2.40(m, 2H, CH2), 3H, 1.85 (t, 1H, J = 2.1 Hz, acetylinic), 1.49 (d, 3H,J = 7.3 Hz, CH3-3), 1.42 (s, 9H, Boc), 1.36 (d, 3H, J = 7.3 Hz, CH3-1),1.25 (d, 6H, J = 13.1 Hz, Me); 13C NMR (CDCl3, 150 MHz): d 170.3,169.3, 155.6, 114.2, 113.0, 106.1, 105.9, 91.6, 89.4, 86.6, 85.5, 85.4,85.2, 82.7, 79.1, 59.1, 59.0, 58.6, 52.5, 44.1, 41.9, 29.7, 26.1, 26.0,25.6, 25.4.; HRMS (ESI+): m/z calculated for C26H42N3O10 (M+ + H)556.2870, found 556.2871.
Boc-L-Ala-b2,3-Caa-L-Ala-b2,3-Caa-OMe (8). To a cooled solutionof 4a (0.08 g, 0.17 mmol), HATU (0.10 g, 0.25 mmol) in dryCH2Cl2 (3 mL) was stirred at 0 1C for 15 min then sequentiallytreated with salt 4b [prepared from 4 (0.08 g, 0.17 mmol) ontreatment with CF3COOH (0.1 mL) in CH2Cl2 (1.5 mL) at 0 1C]and DIPEA (0.06 mL, 0.33 mmol) and stirred for 6 h. Work upas described for 4 and purification of the residue by columnchromatography (60–120 mesh silica gel, 1.5% CH3OH in CHCl3)furnished 8 (0.10 g, 69%) as a white solid; m.p. 112–114 1C; [a]20
D
= +21.6 (c 0.12, CHCl3); IR (KBr): 3358, 3081, 2984, 2929, 2853,1732, 1690, 1524, 1454, 1376, 1323, 1253, 1167, 1117, 1079, 1021,915, 855, 800, 752 cm�1; 1H NMR (600 MHz, CDCl3, 298 K):d 7.97 (d, 1H, J = 9.9 Hz, NH-2), 7.33 (d, 1H, J = 7.2 Hz, NH-3), 6.41(d, 1H, J = 9.9 Hz, NH-4), 5.87 (d, 1H, J = 3.9 Hz, C1H-2), 5.86(d, 1H, J = 3.9 Hz, C1H-4), 5.77 (tdd, 1H, J = 6.5, 10.0, 17.0 Hz,olefinic-2), 5.72 (tdd, 1H, J = 7.5, 10.0, 17.0 Hz, olefinic-4), 5.10(qd, 1H, J = 1.3, 17.0 Hz, olefinic-2), 5.09 (qd, 1H, J = 1.5, 17.0 Hz,olefinic-4), 5.03 (qd, 1H, J = 1.3, 10.0 Hz, olefinic-4), 5.02 (d, 1H,J = 7.0 Hz, NHBoc), 5.0 (qd, 1H, J = 1.3, 10.0 Hz, olefinic-2), 4.56(ddd, 1H, J = 4.0, 6.2, 9.9 Hz, CbH-4), 4.56 (d, 1H, J = 3.9 Hz,C2H-2), 4.55 (d, 1H, J = 3.9 Hz, C2H-4), 4.52 (td, 1H, J = 1.8, 9.9 Hz,CbH-2), 4.44 (qt, 1H, J = 7.2 Hz, CaH-3), 4.12 (dd, 1H, J = 3.3, 9.6Hz, C4H-2), 4.07 (dd, 1H, J = 3.5, 6.2 Hz, C4H-4), 4.02 (qt, 1H,J = 7.0 Hz, CaH-1), 3.88 (d, 1H, J = 3.3 Hz, C3H-2), 3.70 (s, 3H,OMe), 3.65 (d, 1H, J = 3.5 Hz, C3H-4), 3.38 (s, 3H, OMe), 3.33(s, 3H, OMe), 2.63 (ddd, 1H, J = 4.0, 5.2, 9.4 Hz, CaH-4), 2.51(m, 1H, allylic CH2-4), 2.42 (td, 1H, J = 1.8, 7.6 Hz, CaH-2), 2.32(m, 2H, allylic CH2-2), 2.27 (m, 1H, allylic CH2-4), 1.46 (s, 3H, CH3),
1.42 (s, 3H, CH3), 1.41 (d, 3H, J = 7.2 Hz,CH3-3), 1.39 (s, 9H, Boc),1.35 (d, 3H, J = 7.0 Hz, CH3-1), 1.31 (s, 3H, CH3), 1.28 (s, 3H, CH3);13C NMR (150 MHz, CDCl3, 298 K): d 174.4, 173.4, 173.2, 172.7,155.6, 135.2, 134.6, 117.4, 116.9, 111.5, 111.3, 104.8, 104.7, 84.4,83.7, 81.5, 81.2, 80.4, 79.7, 57.4, 57.2, 51.8 (3C), 51.1, 50.0, 49.4,40.1, 48.5, 46.7, 33.5, 33.1, 28.3 (3C), 26.9, 26.7, 26.3 (2C), 17.6,17.4; HRMS (ESI+): m/z (M+ + Na) calculated for C40H64N4O15
863.42604, found 863.42554.
Boc-L-Ala-(S)-b2,3-Caa-L-Ala-(S)-b2,3-Caa-OMe (9). A solution of5a (0.2 g, 0.42 mmol), HOBt (0.07 g, 0.51 mmol) and EDCI(0.1 g, 0.51 mmol) in dry CH2Cl2 (6 mL) was stirred at 0 1C for15 min then sequentially treated with salt 5b [prepared from 5(0.2 g, 0.42 mmol) in anhydrous CH2Cl2 (1 mL) at 0 1C ontreatment with CF3COOH (0.2 mL) in CH2Cl2 (1 mL) at 0 1C]and DIPEA (0.10 mL, 0.63 mmol) and stirred for 6 h. Work up asdescribed for 4 and purification of the residue by columnchromatography (60–120 mesh silica gel, 1.7% CH3OH inCHCl3) afforded 9 (0.26 g, 73%) as a white solid; m.p. 126–128 1C; [a]20
D = �4.35 (c 0.4 in CHCl3); IR (KBr): n 3382, 2983,2934, 1666, 1522, 1451, 1377, 1248, 1167, 1080, 1023, 857,640 cm�1; 1H NMR (CDCl3, 600 MHz): d 7.84 (d, 1H, J = 10.2 Hz,NH-2), 7.39 (d, 1H, J = 7.3 Hz, NH-3), 6.37 (d, 1H, J = 9.8 Hz, NH-4),5.88 (d, 1H, J = 3.7 Hz, C1H-2), 5.86 (d, 1H, J = 3.7 Hz, C1H-4), 5.04(d, 1H, J = 6.6 Hz, NH-1), 4.60 (ddd, 1H, J = 9.8, 9.7, 2.3 Hz, CbH-4),4.58 (d, 1H, J = 3.7 Hz, C2H-2), 4.55 (d, 1H, J = 3.7 Hz, C2H-4), 4.53(ddd, 1H, J = 10.2, 9.8, 2.0 Hz, CbH-2), 4.45 (qd, 1H, J = 8.9, 7.3 Hz,CaH-3), 4.13 (dd, 1H, J = 9.8, 3.2 Hz, C4H-2), 4.12 (dd, 1H, J = 6.2,3.2 Hz, C4H-4), 3.99 (qd, 1H, J = 6.8, 6.6 Hz, CaH-1), 3.92 (d, 1H,J = 3.2, 14.2 Hz, C3H-2), 3.76 (s, 3H, COOMe), 3.69 (d, 1H,J = 3.2 Hz, C3H-4), 3.41 (s, 3H, OMe), 3.34 (s, 3H, OMe), 2.81(ddd, 1H, J = 9.9, 5.1, 2.3 Hz, CaH-4), 2.71 (ddd, 1H, J = 17.0, 9.9, 2.6Hz, CH2-4), 2.66 (ddd, 1H, J = 10.1, 6.2, 2.0 Hz, CaH-2), 2.53 (ddd,1H, J = 16.9, 10.1, 2.6 Hz, CH2-2), 2.44 (ddd, 1H, J = 16.9, 6.2, 2.6Hz, CH2-2), 2.40 (ddd, 1H, J = 17.0, 5.1, 2.6 Hz, CH2-4), 2.20 (t, 1H,J = 1.9 Hz, acetylinic), 1.96 (t, 1H, J = 1.9 Hz, acetylinic), 1.48 (s, 3H,Me), 1.46 (s, 3H, Me), 1.41 (s, 9H, Boc), 1.41 (d, 3H, J = 6.5 Hz, CH3-3), 1.33 (d, 3H, J = 1.9 Hz,CH3-1), 1.31 (s, 3H, Me), 1.29 (s, 3H, Me);13C NMR (150 MHz, CDCl3, 298 K): d 173.5, 173.4, 173.2, 171.3,155.8, 111.6, 111.5, 104.7, 104.7, 84.4, 83.5, 81.4, 81.0, 80.9, 80.0,79.3, 70.3, 69.5, 57.5, 57.4, 52.3, 51.1, 50.1, 49.4, 49.0, 48.4, 46.6,29.7, 28.3(3C), 26.8, 26.7, 26.2, 26.2, 19.0, 19.0, 17.3, 17.1; HRMS(ESI+): m/z calculated for C40H60N4O15 (M+ + Na) 859.3952, found859.3971.
Boc-L-Ala-b3-Caa-L-Ala-b2,3-Caa-OMe (10). A cooled (0 1C)solution of 18 (0.15 g, 0.33 mmol) in THF : MeOH : H2O(3 : 1 : 1) (4 mL) was treated with LiOH (20 mg, 0.84 mmol)and stirred at room temperature. Work up as described for 6gave 18a (0.14 g, 95%) as a colour less solid.
To a cooled solution of 18a (0.09 g, 0.20 mmol), HOBt(0.04 g, 0.31 mmol) and EDCI (0.06 g, 0.31 mmol) in dry CH2Cl2
(4 mL) was stirred at 0 1C for 15 min and treated sequentiallywith the salt 4b [prepared from 4 (0.10 g, 0.20 mmol) andCF3COOH (0.1 mL) in CH2Cl2 (1 mL) at 0 1C] and DIPEA(0.07 mL, 0.41 mmol) and stirred at room temperature for
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
6 h. Work up as described for 4 and purification of the residueby column chromatography (60–120 mesh silica gel, 1.6%CH3OH in CHCl3) furnished 10 (0.12 g, 72%) as a white solid;m.p. 111–113 1C; [a]20
D = �6.20 (c 0.21, CHCl3); IR (KBr): 3334,2984, 2934, 1664, 1527, 1452, 1376, 1218, 1168, 1117, 1080,1022, 857, 637 cm�1; 1H NMR (600 MHz, CDCl3, 298 K): d 7.59(d, 1H, J = 9.0 Hz, NH-2), 7.47 (d, 1H, J = 7.1 Hz, NH-3), 6.51(d, 1H, J = 9.7 Hz, NH-4), 5.89 (d, 1H, J = 3.8 Hz, C1H-4), 5.87(d, 1H, J = 3.8 Hz, C1H-2), 5.73 (tdd, 1H, J = 7.5, 10.0, 17.0 Hz,olefinic), 5.09 (qd, 1H, J = 1.5, 17.0 Hz, olefinic), 5.07 (d, 1H,J = 6.4 Hz, NHBoc), 5.03 (qd, 1H, J = 1.3, 10.0 Hz, olefinic), 4.56(ddd, 1H, J = 4.0, 6.2, 9.9 Hz, CbH-4), 4.56 (d, 1H J = 3.8 Hz,C2H-2), 4.55 (d, 1H, J = 3.8 Hz, C2H-4), 4.49 (m, 1H, CbH-2), 4.40(qt, 1H, J = 7.0 Hz, C1H-3), 4.24 (dd, 1H, J = 3.0, 9.4 Hz, C4H-2),4.11 (dd, 1H, J = 3.5, 6.2 Hz, C4H-4), 4.06 (dq, 1H, J = 6.4, 7.0 Hz,CaH-1), 3.97 (d, 1H, J = 3.0 Hz, C3H-2), 3.70 (s, 3H, OMe), 3.65(d, 1H, J = 3.5 Hz, C3H-4), 3.38 (s, 3H, OMe), 3.33 (s, 3H, OMe),2.63 (ddd, 1H, J = 3.8, 5.7, 9.4 Hz, CaH-4), 2.56 (dd, 1H, J = 5.3,13.3 Hz, CaH-2), 2.49 (m, 1H, allylic CH2), 2.30 (m, 2H, allylicCH2 and CaH-2), 1.46 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.40 (s, 9H,Boc), 1.37 (d, 3H, J = 7.2 Hz, CH3-3), 1.34 (d, 3H, J = 7.0 Hz,CH3-1), 1.30 (s, 3H, CH3), 1.29 (s, 3H, CH3); 13C NMR (150 MHz,CDCl3, 298 K): d 174.4, 173.2, 173.1, 170.6, 155.8, 134.7, 117.4,111.5, 111.4, 104.9, 104.7, 84.3, 83.5, 81.5, 81.1, 79.9, 79.8, 79.6,57.4, 57.3, 51.9, 51.0, 49.9, 48.5, 46.7, 46.6, 38.4, 33.4, 28.2 (3C),26.8, 26.7, 26.3, 26.2, 17.6, 17.3; HRMS (ESI+): m/z (M+ + Na)calculated for C37H60N4O15 823.39474, found 823.39416.
Boc-L-Ala-b2,3-Caa-L-Ala-b3-Caa-OMe (11). A cooled (0 1C)solution of 4a (0.06 g, 0.12 mmol), HOBt (25 mg, 0.19 mmol)and EDCI (36 mg, 0.19 mmol) in dry CH2Cl2 (3 mL) was stirredat 0 1C for 15 min and treated sequentially with the salt 18b[prepared from 18 (56 mg, 0.12 mmol) and CF3COOH (0.1 mL)in CH2Cl2 (1 mL)] and DIPEA (0.04 mL, 0.25 mmol) and stirredat room temperature for 6 h. Work up as described for 4 andpurification of the resude by column chromatography (60–120mesh silica gel, 1.7% CH3OH in CHCl3) furnished 11 (0.07 g,70%) as a white solid; m.p. 108–110 1C; [a]20
D = +0.38 (c 0.26,CHCl3); IR (KBr): 3332, 2984, 2935, 1726, 1691, 1654, 1525,1454, 1373, 1249, 1211, 1168, 1120, 1080, 1022, 910, 856,787 cm�1; 1H NMR (600 MHz, CDCl3, 298 K): d 7.90 (d, 1H,J = 10.2 Hz, NH-2), 7.38 (d, 1H, J = 7.4 Hz, NH-3), 6.49 (d, 1H, J =8.2 Hz, NH-4), 5.92 (d, 1H, J = 3.8 Hz, C1H-4), 5.77 (tdd, 1H, J =7.0, 10.0, 17.0 Hz, olefinic), 5.76 (d, 1H, J = 3.8 Hz, C1H-2), 5.09(qd, 1H, J = 1.5, 17.0 Hz, olefinic), 5.0 (m, 2H, olefinic andNHBoc), 4.57 (d, 1H, J = 3.8 Hz, C2H-2), 4.56 (d, 1H, J = 3.8 Hz,C2H-4), 4.53 (td, 1H, J = 1.7, 10.2 Hz, CbH-2), 4.49 (m, 1H,CbH-4), 4.39 (m, 2H, CaH-3 and C4H-4), 4.10 (dd, 1H, J = 3.1,9.8 Hz, C4H-2), 4.01 (qt, 1H, J = 6.3 Hz, CaH-1), 3.91 (d, 1H, J =3.1 Hz, C3H-2), 3.69 (s, 3H, OMe), 3.67 (d, 1H, J = 3.4 Hz, C3H-4),3.38 (s, 3H, OMe), 3.36 (s, 3H, OMe), 2.68 (dd, 1H, J = 6.6,16.5 Hz, CaH-4), 2.61 (dd, 1H, J = 4.8, 16.5 Hz, CaH-4), 2.43(m, 1H, CaH-2), 2.31 (m, 2H, allylic CH2), 1.48 (s, 3H, CH3), 1.43(s, 3H, CH3), 1.41 (d, 3H, J = 7.4 Hz, CH3-3), 1.39 (s, 9H, Boc),1.34 (d, 3H, J = 6.38 Hz, CH3-1), 1.31 (s, 3H, CH3), 1.29 (s, 3H,CH3); 13C NMR (150 MHz, CDCl3, 298 K): d 173.4, 173.2, 172.9,
171.9, 155.7, 135.2, 116.9, 111.6, 111.3, 104.9, 104.7, 84.4, 83.6,81.5, 81.3, 80.3, 79.8, 78.9, 57.5, 57.3, 51.8, 51.2, 49.8 (2C), 48.9,45.7, 35.9, 33.0, 28.2 (3C), 26.7 (2C), 26.4, 26.2, 17.3, 16.8; HRMS(ESI+): m/z (M+ + Na) calculated for C37H60N4O15 823.39474,found 823.39461.
Boc-L-Ala-(S)-b2,3-Caa-L-Ala-OMe-(S)-b2,3-Caa-L-Ala-OMe (12).To a solution of 5a (0.09 g, 0.16 mmol), HOBt (0.03 g,0.19 mmol) and EDCI (0.04 g, 0.19 mmol) in dry CH2Cl2
(3 mL), salt 7a [prepared from 7 (0.08 g, 0.16 mmol) andCF3COOH (0.1 mL) in CH2Cl2 (1 mL)] was added and stirredat room temperature for 5 h. Work up as described for 4 andpurification of the residue by column chromatography (60–120mesh silica gel, 2.1% CH3OH in CHCl3) furnished 12 (0.08 g,58%) as a white solid; m.p. 136–138 1C; [a]20
D = +48.9 (c 0.38,CHCl3); IR (KBr): n 3353, 2985, 2936, 1667, 1519, 1454, 1377,1241, 1165, 1079, 1022, 856, 639 cm�1; 1H NMR (CDCl3,500 MHz): d 7.91 (d, 1H, J = 9.5 Hz, NH-2), 7.63 (d, 1H, J =7.3 Hz, NH-5), 7.57 (d, 1H, J = 5.4 Hz, NH-3), 7.41 (d, 1H, J =10.2 Hz, NH-4), 5.86 (d, 1H, J = 3.7 Hz, C1H-2), 5.85 (d, 1H, J =3.7 Hz, C1H-4), 5.11 (d, 1H, J = 6.5 Hz, NH-1), 4.58 (d, 1H, J =3.7 Hz, C2H-2), 4.58 (dd, 1H, J = 7.3, 7.1 Hz, CaH-5), 4.56 (d, 1H,J = 3.7 Hz, C2H-4), 4.49 (dd, 1H, J = 9.5, 2.9 Hz, CbH-2), 4.43 (dd,1H, J = 9.8, 1.8 Hz, CbH-4), 4.17 (qd, 1H, J = 5.4, 7.4 Hz, CaH-3),4.14 (dd, 1H, J = 9.3, 3.2 Hz, C4H-2), 4.13 (dd, 1H, J = 9.8, 3.2 Hz,C4H-4), 4.03 (qd, 1H, J = 6.5, 7.0 Hz, CaH-1), 3.89 (d, 1H,J = 3.2 Hz, C3H-4), 3.86 (d, 1H, J = 3.2 Hz, C3H-2), 3.74 (s, 3H,COOMe), 3.42 (s, 3H, OMe), 3.41 (s, 3H, OMe), 2.70 (ddd, 1H,J = 16.9, 10.8, 2.5 Hz, CH2-4), 2.66 (ddd, 1H, J = 10.0, 5.9, 2.9 Hz,CaH-2), 2.64 (ddd, 1H, J = 10.8, 4.8, 1.8 Hz, CaH-4), 2.53 (ddd,1H, J = 17.1, 10.1, 2.5 Hz, CH2-2), 2.48 (ddd, 1H, J = 18.1, 5.9,2.5 Hz, CH2-2), 2.41 (ddd, 1H, J = 16.9, 4.8, 2.5 Hz, CH2-4), 1.96(s, 1H, acetylinic), 1.90 (s, 1H, acetylinic), 1.52 (s, 3H, Me), 1.48(d, 3H, J = 7.4 Hz, CH3-3), 1.47 (d, 3H, J = 7.4 Hz, CH3-5), 1.45(d, 3H, J = 7.1 Hz, Me), 1.42 (s, 9H, Boc), 1.33 (d, 3H, J = 7.0 Hz,CH3-1), 1.30 (s, 6H, Me); 13C NMR (150 MHz, CDCl3, 278 K):d 174.3, 173.6, 171.9, 171.4, 169.8, 155.8, 111.5, 104.7, 104.6,196.1, 183.6, 81.3, 81.3, 81.2, 80.1, 79.8, 79.6, 69.8, 69.4, 57.4,57.3, 52.6, 51.5, 51.1, 49.7, 49.7, 49.6, 49.4, 49.3, 48.9, 48.8, 28.3,26.8, 26.7, 26.4, 26.1, 19.2, 19.0, 17.0, 16.7, 16.5.; HRMS (ESI+):m/z calculated for C43H65N5O16 (M+ + Na) 930.4324, found930.4362.
Boc-L-Ala-b3-Caa-L-Ala-b2,3-Caa-L-Ala-OMe (13a). A cooled(0 1C) solution of 18a (0.05 g, 0.11 mmol), HOBt (23 mg,0.17 mmol) and EDCI (33 mg, 0.17 mmol) in dry CH2Cl2
(3 mL) was stirred at 0 1C for 15 min and treated sequentiallywith the salt 6a [prepared from 6 (0.06 g, 0.11 mmol) andCF3COOH (0.1 mL) in CH2Cl2 (1 mL)] and DIPEA (0.04 mL,0.23 mmol) and stirred at room temperature for 6 h. Work up asdescribed for 4 and purification of the residue by columnchromatography (60–120 mesh silica gel, 2.2% CH3OH inCHCl3) furnished 13a (63 mg, 63%) as a white solid; m.p.125–127 1C; [a]20
D = +27.3 (c 0.17, CHCl3); IR (KBr): 3336, 2985,2933, 1663, 1532, 1456, 1377, 1323, 1220, 1167, 1118, 1079,1022, 856 cm�1; 1H NMR (600 MHz, CDCl3, 298 K): d 7.99
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
(d, 1H, J = 10.2 Hz, NH-2), 7.92 (d, 1H, J = 7.5 Hz, NH-5), 7.71(d, 1H, J = 6.1 Hz, NH-3), 7.23 (d, 1H, J = 9.2 Hz, NH-4), 5.88 (d, 1H,J = 4.0 Hz, C1H-2), 5.87 (d, 1H, J = 4.0 Hz, C1H-4), 5.77 (m, 1H,olefinic), 5.11 (m, 1H, olefinic), 5.01 (m, 1H, olefinic), 4.95 (d, 1H,J = 6.2 Hz, NH-1), 4.60 (d, 1H, J = 4.0 Hz, C2H-2), 4.56 (q, 1H,J = 7.5 Hz, CaH-5), 4.55 (d, 1H, J = 4.0 Hz, C2H-4), 4.54 (td, 1H,J = 1.8, 10.2 Hz, CbH-2), 4.43 (tdd, 1H, J = 2.8, 5.1, 10.2 Hz, CbH-4),4.20 (dd, 1H, J = 3.7, 10.2 Hz, C4H-2), 4.15 (dd, 1H, J = 3.3, 9.6 Hz,C4H-4), 4.13 (qd, 1H, J = 6.1, 7.5 Hz, CaH-3), 4.03 (d, 1H, J = 3.3,4.0 Hz, C3H-4), 4.01 (qd, 1H, J = 6.2, 7.1 Hz, CaH-1), 3.84 (d, 1H J =3.3 Hz, C3H-2), 3.72 (s, 3H, OCH3), 3.39 (s, 3H, OCH3), 3.37 (s, 3H,OCH3), 2.62 (dd, 1H, J = 5.1, 12.5 Hz, CaH-2), 2.39 (m, 1H, CaH-4),2.35 (m, 2H, CH2), 2.13 (dd, 1H, J = 2.6, 12.5 Hz, CaH-2), 1.49 (d,3H, J = 7.5 Hz, CH3-3), 1.48 (s, 3H, CH3), 1.45 (d, 3H, J = 7.5 Hz,CH3-5), 1.43 (s, 3H, CH3), 1.39 (s, 9H, Boc), 1.35 (d, 3H, J = 7.0 Hz,CH3-1), 1.31 (s, 3H, CH3), 1.28 (s, 3H, CH3); 13C NMR (75 MHz,CDCl3, 298 K): d 175.6, 174.9, 174.1, 173.6, 170.8, 155.7, 135.1,116.9, 111.7, 111.2, 105.0, 104.9, 83.7, 83.4, 81.5, 81.3, 80.6, 79.9,79.7, 57.3, 57.2, 52.5, 52.1, 51.5, 49.8, 49.2, 48.7, 47.2, 38.2, 32.4,28.3 (3C), 26.8, 26.7, 26.4, 26.0, 17.1, 16.3, 16.1; HRMS (ESI+): m/z(M+ + Na) calculated for C40H65N5O16 894.43185, found 894.43167.
Boc-L-Ala-b2,3-Caa-L-Ala-b2,3-Caa-L-Ala-b3-Caa-OMe (14). To acooled solution of 4a (0.13 g, 0.06 mmol), HATU (36 mg,0.09 mmol) in dry CH2Cl2 (2 mL) was stirred at 0 1C for15 min and treated sequentially with the salt 11a [preparedfrom 11 (0.05 g, 0.06 mmol) and CF3COOH (0.1 mL) in CH2Cl2
(1 mL)] and DIPEA (0.02 mL, 012 mmol) and stirred at roomtemperature for 6 h. Work up as described for 4 and purifica-tion of the residue by column chromatography (60–120 meshsilica gel, 2.9% CH3OH in CHCl3) furnished 14 (47 mg, 64%) asa white solid; m.p. 138–140 1C; [a]20
D = +11.3 (c 0.25, CHCl3); IR(KBr): 3351, 2963, 2931, 1653, 1524, 1455, 1378, 1261, 1166,1083, 1022, 859, 802, 664 cm�1; 1H NMR (600 MHz, CDCl3,298 K): d 7.77 (d, 1H, J = 10.0 Hz, NH-2), 7.68 (d, 1H, J = 10.1 Hz,NH-4), 7.30 (d, 1H, J = 6.2 Hz, NH-3), 7.14 (d, 1H, J = 7.3 Hz,NH-5), 6.46 (d, 1H, J = 8.2 Hz, NH-6), 5.84 (d, 1H, J = 3.8 Hz,C1H-4), 5.79 (d, 1H, J = 3.8 Hz, C1H-6), 5.78 (d, 1H, J = 3.8 Hz,C1H-2), 5.69 (m, 1H, olefinic), 5.67 (m, 1H, olefinic), 5.09(m, 1H, olefinic), 5.01 (m, 1H, olefinic), 4.98 (m, 2H, olefinicand NHBoc), 4.92 (m, 1H, olefinic), 4.50 (d, 1H, J = 3.8 Hz,C2H-6), 4.49 (d, 1H, J = 3.8 Hz, C2H-4), 4.46 (d, 1H, J = 3.8 Hz,C2H-2), 4.45 (m, 1H, CbH-2), 4.44 (ddd, 1H, J = 5.9, 6.3, 8.2 Hz,CbH-6), 4.40 (m, 1H, CbH-4), 4.40 (dq, 1H, J = 7.2, 7.3 Hz,CaH-5), 4.31 (dd, 1H, J = 3.3, 6.5 Hz, C4H-6), 4.23 (dq, 1H, J = 6.2,7.6 Hz, CaH-3), 4.13 (dd, 1H, J = 3.4, 9.4 Hz, C4H-2), 4.06 (dd,1H, J = 3.3, 9.5 Hz, C4H-4), 4.02 (dq, 1H, J = 6.4, 7.0 Hz, CaH-1),3.76 (d, 1H, J = 3.3 Hz, C3H-4), 3.74 (d, 1H, J = 3.4 Hz, C3H-2),3.62 (s, 3H, OMe), 3.60 (d, 1H, J = 3.3 Hz, C3H-6), 3.31 (s, 3H,OMe), 3.30 (s, 6H, 2 � OMe), 2.63 (dd, 1H, J = 6.3, 16.4 Hz,CaH-6), 2.52 (dd, 1H, J = 5.9, 16.4 Hz, CaH-6), 2.38-2.22 (m, 6H,2 � allylic CH2, 2 � CaH), 1.41 (s, 3H, CH3), 1.38 (s, 3H, CH3),1.38 (d, 3H, J = 7.6 Hz, CH3-3), 1.36 (s, 3H, CH3), 1.33 (s, 9H,Boc), 1.31 (d, 3H, J = 7.2 Hz, CH3-5), 1.25 (d, 3H, J = 7.0 Hz,CH3-1), 1.25 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.20 (s, 3H, CH3);13C NMR (150 MHz, CDCl3, 298 K): d 174.2, 173.6, 173.2, 173.0,
172.9, 171.8, 155.6, 135.1, 134.9, 117.3, 116.9, 111.7, 111.3,104.8, 104.7, 84.4, 83.7, 83.6, 81.4, 81.3, 81.2, 80.2, 80.1, 79.7,79.0, 57.5, 57.3, 51.9, 51.1 (2C), 49.9, 49.5, 49.4, 49.2, 49.1, 45.6,35.9, 33.4, 33.3, 29.7, 29.3, 28.2 (3C), 26.8, 26.7, 26.6, 26.3, 26.2,26.1, 22.7, 17.3, 17.2, 17.0; HRMS (ESI+): m/z (M+ + Na)calculated for C54H86N6O21 1177.57382, found 1177.57261.
Boc-L-Ala-(S)-b2,3-Caa-L-Ala-OMe-(S)-b2,3-Caa-L-Ala-OMe-(S)-b3-Caa-OMe (15). A solution of 9 (0.1 g, 0.18 mmol) in MeOH(2 mL) was treated with aq. 4 N NaOH solution (2 mL) at 0 1C toroom temperature for 1 h. Work up as described for 4a gave 9a(0.09 g, 92%) as a colorless solid.
To a solution of 9a (0.09 g, 0.12 mmol), HOBt (0.02 g,0.14 mmol) and EDCI (0.03 g, 0.14 mmol) in dry CH2Cl2
(3 mL), salt 18b [prepared from 18 (0.05 g, 0.12 mmol) andCF3COOH (0.05 mL) in CH2Cl2 (0.5 mL) at 0 1C] was added andstirred at room temperature for 6 h. Work up as described for 4and purification of the residue by column chromatography(60–120 mesh silica gel, 3.5% CH3OH in CHCl3) furnished 15(0.09 g, 62%) as a white solid; m.p. 141–144 1C; [a]20
D = +43.48(c 0.22, CHCl3); IR (KBr): n 3373, 2928, 2856, 1731, 1657, 1524,1452, 1376, 1249, 1166, 1080, 1022, 858, 635 cm�1; 1H NMR(CDCl3, 500 MHz): d 7.66 (d, 1H, J = 9.8 Hz, NH-2), 7.60 (d, 1H,J = 9.2 Hz, NH-4), 7.40 (d, 1H, J = 7.0 Hz, NH-3), 7.17 (d, 1H,J = 5.1 Hz, NH-5), 6.52 (d, 1H, J = 9.6 Hz, NH-6), 5.91 (d, 1H,J = 3.7 Hz, C1H-6), 5.87 (d, 1H, J = 3.7 Hz, C1H-4), 5.85 (d, 1H,J = 3.7 Hz, C1H-2), 5.11 (d, 1H, J = 7.0 Hz, NH-1), 4.57 (d, 1H,J = 3.7 Hz, C2H-6), 4.57 (d, 1H, J = 3.7 Hz, C2H-4), 4.56 (d, 1H,J = 3.7 Hz, C2H-2), 4.52 (ddd, 1H, J = 10.2, 10.8, 2.3 Hz, CbH-2),4.41 (ddd, 1H, J = 8.8, 6.2, 5.2 Hz, CbH-6), 4.49 (ddd, 1H, J = 9.7,8.8, 2.3 Hz, CbH-4), 4.39 (dd, 1H, J = 6.1, 3.5 Hz, C4H-6), 4.38(qd, 1H, J = 7.3, 7.0 Hz, CaH-5), 4.32 (dd, 1H, J = 7.3, 5.8 Hz,CaH-3), 4.17 (dd, 1H, J = 8.8, 3.4 Hz, C4H-2), 4.14 (dd, 1H,J = 8.8, 3.0 Hz, C4H-4), 4.03 (qd, 1H, J = 7.1, 6.6 Hz, CaH-1), 3.87(d, 1H, J = 3.4 Hz, C3H-2), 3.80 (d, 1H, J = 3.0 Hz, C3H-4), 3.69(s, 3H, COOMe), 3.60 (d, 1H, J = 3.4 Hz, C3H-6), 3.40 (s, 6H, 2 �OMe), 3.37 (s, 3H, OMe), 2.69 (ddd, 1H, J = 17.0, 5.5, 2.5 Hz,CH2-2), 2.69 (ddd, 1H, J = 10.1, 5.8, 1.9 Hz, CaH-4), 2.69 (dd, 1H,J = 16.1, 5.2 Hz, CaH-6), 2.67 (ddd, 1H, J = 10.1, 5.0, 3.0 Hz,CaH-2), 2.65 (ddd, 1H, J = 17.0, 11.0, 2.5 Hz, CH2-4), 2.60 (dd,1H, J = 16.1, 6.2 Hz, CaH-6), 2.46 (ddd, 1H, J = 17.0, 4.9, 2.5 Hz,CH2-4), 2.41 (ddd, 1H, J = 17.1, 9.6, 2.5 Hz, CH2-2), 2.03 (t, 1H,J = 1.9 Hz, acetylinic), 1.95 (s, 1H, J = 1.9 Hz, acetylinic), 1.50(s, 3H, Me), 1.48 (s, 6H, Me), 1.42 (s, 9H, Boc), 1.44 (d, 3H,J = 7.3 Hz, CH3-5), 1.42 (s, 9H, Boc), 1.39 (d, 3H, J = 7.3 Hz,CH3-3), 1.32 (d, 3H, J = 7.3 Hz, CH3-1), 1.31 (s, 3H, Me), 1.29(s, 3H, Me), 1.25 (s, 3H, Me); 13C NMR (150 MHz, CDCl3, 278 K):d 173.7, 173.4, 173.2, 173.1, 171.5, 171.0, 155.9, 111.6, 111.6, 111.4,104.9, 104.7, 104.7, 84.4, 83.7, 81.7, 81.4, 81.3, 81.1, 80.8, 80.0, 79.8,79.3, 70.3, 70.3, 70.2, 57.5, 57.4, 57.2, 52.2, 51.0, 50.8, 50.0, 49.8,48.6, 48.5, 46.6, 46.5, 38.1, 29.7, 28.2 (3C), 26.9, 26.8, 26.7, 26.2,26.2, 19.5, 19.0, 17.6, 17.4, 16.9.; HRMS (ESI+): m/z calculated forC54H82N6O21 (M+ + Na) 1173.5430, found 1173.5427.
Boc-L-Ala-b2,3-Caa-L-Ala-b3-Caa-L-Ala-b2,3-Caa-OMe (16). A cooled(0 1C) solution of 4a (0.03 g, 0.06 mmol), HATU (36 mg, 0.09 mmol)
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
New J. Chem. This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015
in dry CH2Cl2 (2 mL) was stirred at 0 1C for 15 min and treatedsequentially with the salt 10a [prepared from 10 (0.05 g, 0.06 mmol)and CF3COOH (0.1 mL) in CH2Cl2 (1 mL)] and DIPEA (0.02 mL,0.12 mmol) and stirred at room temperature for 6 h. Work upas described for 4 and purification of the residue by columnchromatography (60–120 mesh silica gel, 2.8% CH3OH in CHCl3)furnished 16 (45 mg, 62%) as a white solid; m.p. 142–144 1C; [a]20
D =+20.2 (c 0.11, CHCl3); IR (KBr): 3345, 2929, 1652, 1536, 1451, 1377,1249, 1167, 1080, 1023, 857 cm�1; 1H NMR (600 MHz, CDCl3,298 K): d 8.12 (d, 1H, J = 10.5 Hz, NH-2), 7.89 (d, 1H, J = 9.3 Hz,NH-4),7.85 (d, 1H, J = 7.2 Hz, NH-5), 7.65 (d, 1H, J = 6.0 Hz, NH-3),6.56 (d, 1H, J = 9.7 Hz, NH-6), 5.90 (d, 1H, J = 3.8 Hz, C1H-2), 5.87(d, 1H, J = 3.2 Hz, C1H-4), 5.86 (d, 1H, J = 3.6 Hz, C1H-6), 5.75(m, 1H, olefinic), 5.72 (m, 1H, olefinic), 5.10 (m, 2H, olefinic), 5.02(m, 1H, olefinic), 4.99 (m, 1H, olefinic), 4.97 (d, 1H, J = 6.4 Hz,NHBoc), 4.57 (d, 1H, J = 3.2 Hz, C2H-4), 4.55 (m, 3H, 2 � C2H andCbH-6), 4.53 (td, 1H, J = 1.5, 10.5 Hz, CbH-2), 4.41 (m, 1H, CbH-4),4.39 (qt, 1H, J = 7.2 Hz, CaH-5), 4.26 (dd, 1H, J = 3.2, 10.1 Hz,C4H-4), 4.24 (qt, 1H, J = 6.3 Hz, CaH-3), 4.15 (dd, 1H, J = 3.3, 9.9 Hz,C4H-2), 4.11 (dd, 1H, J = 3.3, 6.4 Hz, C4H-6), 4.02 (dq, 1H,J = 6.4, 7.0 Hz, CaH-1), 4.02 (d, 1H, J = 3.2 Hz, C3H-4), 3.86(d, 1H, J = 3.3 Hz, C3H-2), 3.70 (s, 3H, OMe), 3.66 (d, 1H, J = 3.3 Hz,C3H-6), 3.38 (s, 3H, OMe), 3.37 (s, 3H, OMe), 3.32 (s, 3H, OMe),2.64 (ddd, 1H, J = 3.9, 5.2, 9.4 Hz, CaH-6), 2.58 (dd, 1H, J = 5.2,13.1 Hz, CaH-4), 2.50 (m, 1H, allylic CH2-6), 2.39–2.34 (m, 3H,allylic CH2), 2.16 (m, 2H, allylic CH2), 1.46 (s, 3H, CH3), 1.45 (s, 3H,CH3), 1.44 (s, 3H, CH3), 1.40 (m, 3H, CH3-5), 1.39 (s, 9H, Boc), 1.37(d, 3H, J = 6.3 Hz, CH3-3), 1.34 (d, 3H, J = 7.0 Hz, CH3-1), 1.30 (s,3H, CH3), 1.29 (s, 6H, 2� CH3); 13C NMR (150 MHz, CDCl3, 298 K):d 175.2, 174.5, 173.9, 173.6 (2C), 170.6, 155.6, 135.1, 134.7, 117.3,116.6, 111.5, 111.2, 105.0, 104.9, 84.4, 83.6, 83.4, 81.5, 51.2, 81.2,80.7, 79.8, 79.5, 57.5, 57.3, 57.2, 51.9, 51.9, 51.8, 51.5, 50.2, 50.7,49.2, 48.4, 47.3, 46.9, 38.3, 33.5, 32.4, 31.9, 29.7, 28.3 (3C), 26.9,26.7, 26.6, 26.2 (2C), 26.1, 17.1, 16.9, 16.5; HRMS (ESI+): m/z (M+ +Na) calculated for C54H86N6O21 1177.57382, found 1177.57499.
Boc-L-Ala-(S)-b3-Caa-L-Ala-OMe-(S)-b2,3-Caa-L-Ala-OMe-(S)-b2,3-Caa-OMe (17). A solution of 18a (0.12 g, 0.25 mmol), HOBt(0.04 g, 0.3 mmol) and EDCI (0.06 g, 0.3 mmol) in dry CH2Cl2
(3 mL), amine salt 9b [prepared from 9 (0.21 g, 0.25 mmol) andCF3COOH (0.2 mL) in CH2Cl2 (2 mL)] was added and stirredat room temperature for 5 h. Work up as described for 4and purification of the residue by column chromatography(60–120 mesh silica gel, 3.1% CH3OH in CHCl3) furnished 17(0.19 g, 67%) as a white solid; m.p. 144–148 1C; [a]20
D = +51.38(c 0.24, CHCl3); IR (KBr): n 3373, 2928, 2856, 1731, 1657, 1524,1452, 1376, 1249,1166, 1080, 1022, 858, 635 cm�1; 1H NMR(CDCl3, 500 MHz): d 7.76 (d, 1H, J = 9.8 Hz, NH-4), 7.61 (d, 1H,J = 9.2 Hz, NH-2), 7.43 (d, 1H, J = 7.0 Hz, NH-5), 7.38 (d, 1H,J = 5.1 Hz, NH-3), 6.43 (d, 1H, J = 9.6 Hz, NH-6), 5.87 (d, 1H,J = 3.7 Hz, C1H-2), 5.87 (d, 1H, J = 3.7 Hz, C1H-4), 5.86 (d, 1H,J = 3.7 Hz, C1H-6), 5.31 (d, 1H, J = 7.0 Hz, NH-1), 4.60 (ddd, 1H,J = 9.6, 9.2, 3.6 Hz, CbH-6), 4.58 (d, 1H, J = 3.7 Hz, C2H-4), 4.56(d, 1H, J = 3.7 Hz, C2H-2), 4.55 (d, 1H, J = 3.7 Hz, C2H-6), 4.45(t, 1H, J = 9.8, 1.9 Hz, CbH-4), 4.44 (ddd, 1H, J = 9.2, 8.1, 2.7 Hz,CbH-2), 4.42 (qd, 1H, J = 7.3, 7.0 Hz, CaH-5), 4.28 (dd, 1H,
J = 9.2, 3.1 Hz, C4H-2), 4.19 (qd, 1H, J = 7.3, 5.1 Hz, CaH-3), 4.14(dd, 1H, J = 9.8, 3.0 Hz, C4H-4), 4.13 (dd, 1H, J = 6.4, 3.0 Hz,C4H-6), 4.08 (dq, 1H, J = 7.3, 7.0 Hz, CaH-1), 3.92 (d, 1H,J = 3.0 Hz, C3H-2), 3.86 (d, 1H, J = 3.0 Hz, C3H-4), 3.76 (s, 3H,COOMe), 3.68 (d, 1H, J = 3.0 Hz, C3H-6), 3.40 (s, 3H, OMe), 3.38(s, 3H, OMe), 3.34 (s, 3H, OMe), 2.80 (ddd, 1H, J = 9.6, 3.6,5.5 Hz, CaH-6), 2.69 (ddd, 1H, J = 17.0, 5.5, 2.5 Hz, CH2-6), 2.66(dd, 1H, J = 4.2, 13.2 Hz, CaH-2), 2.65 (ddd, 1H, J = 17.0, 5.5, 2.5Hz, CH2-4), 2.59 (ddd, 1H, J = 11.0, 4.9, 1.9 Hz, CaH-4), 2.46(ddd, 1H, J = 17.1, 4.9, 2.5 Hz, CH2-4), 2.41 (ddd, 1H, J = 17.1,9.6, 2.5 Hz, CH2-6), 2.25 (dd, 1H, J = 13.2, 4.2 Hz, CaH-2), 2.01(t, 1H, J = 1.9 Hz, acetylinic), 2.00 (s, 1H, J = 1.9 Hz, acetylinic),1.52 (s, 3H, Me), 1.46 (s, 6H, Me), 1.46 (s, 3H, Me), 1.42 (s, 9H,Boc), 1.38 (d, 3H, J = 7.3 Hz, CH3-5), 1.35 (d, 3H, J = 7.3 Hz,CH3-3), 1.31 (d, 3H, J = 7.3 Hz, CH3-1), 1.30 (s, 3H, Me), 1.25(s, 3H, Me); 13C NMR (150 MHz, CDCl3, 278 K): d 173.9, 173.4,173.4, 173.1, 171.5, 171.0, 155.8, 111.6, 111.6, 111.4, 104.8, 104.7,104.7, 96.1, 84.3, 83.6, 83.5, 81.4, 81.2, 81.0, 80.8, 79.9, 79.8, 79.7,79.3, 70.3, 70.1, 57.5, 57.4, 57.2, 52.3, 51.1, 50.8, 50.1, 49.8, 48.6,48.4, 46.8, 46.5, 38.0, 29.7, 28.2 (3C), 26.8, 26.7, 26.7, 26.2, 26.1,19.5, 19.0, 17.5, 17.4, 16.9; HRMS (ESI+): m/z calculated forC54H82N6O21 (M+ + Na) 1173.5430, found 1173.5423.
Acknowledgements
The authors are thankful for financial support from the Councilof Scientific and Industrial Research (CSIR), New Delhi (CSC-0114) and to Dr A. Ravi Sankar for several fruitful discussions.T. S. R., B. V. and P. P. R. are thankful to CSIR, New Delhi, forfinancial support in the form of fellowships.
Notes and references
1 (a) D. J. Hill, M. J. Mio, R. B. Prince, T. S. Hughes and J. S.Moore, Chem. Rev., 2001, 101, 3893–4011; (b) J. Venkatraman,S. C. Shankaramma and P. Balaram, Chem. Rev., 2001, 101,3131–3152.
2 (a) D. Seebach and J. L. Matthews, Chem. Commun., 1997,2015–2022; (b) S. H. Gellman, Acc. Chem. Res., 1998, 31,173–180; (c) K. Kirschenbaum, R. N. Zuckerman andD. A. Dill, Curr. Opin. Struct. Biol., 1999, 9, 530–535;(d) K. D. Stigers, M. J. Soth and J. S. Nowick, Curr. Opin.Chem. Biol., 1999, 3, 714–723; (e) M. D. Smith and G. W. J.Fleet, J. Pept. Sci., 1999, 5, 425–441; ( f ) R. P. Cheng,S. H. Gellman and W. F. De Grado, Chem. Rev., 2001, 101,3219–3232; (g) M. S. Cubberley and B. L. Iverson, Curr. Opin.Chem. Biol., 2001, 5, 650–653; (h) F. Fulop, Chem. Rev., 2001,101, 2181–2204; (i) D. Seebach, D. F. Hook and A. Glattli,Biopolymers, 2005, 84, 23–37; ( j) T. A. Martinek and F. Fulop,Eur. J. Biochem., 2003, 270, 3657–3666; (k) N. Rathore,S. H. Gellman and J. J. de Pablo, Biophys. J., 2006, 91,3425–3435; (l) C. M. Goodman, S. Choi, S. Shandler andW. F. DeGrado, Nat. Chem. Biol., 2007, 3, 252–262;(m) S. Hecht and I. Huc, in Foldamers: Structure, Propertiesand Applications, ed. S. Hecht and I. Huc, Wiley-VCH,
Paper NJC
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
This journal is©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2015 New J. Chem.
Weinheim, Germany, 2007; (n) I. Saraogi and A. D. Hamilton,Chem. Soc. Rev., 2009, 38, 1726–1743; (o) G. Guichard andI. Huc, Chem. Commun., 2011, 5933–5941; (p) P. G. Vasudev,S. Chatterjee, N. Shamala and P. Balaram, Chem. Rev., 2011,111, 657–687; (q) F. Bouillere, S. Thetiot-Laurent, C. Kouklovskyand V. Alezra, Amino Acids, 2011, 41, 687–707; (r) T. A. Martinekand F. Fulop, Chem. Soc. Rev., 2012, 41, 687–702.
3 (a) D. H. Appella, L. A. Christianson, I. L. Karle, D. R. Powelland S. H. Gellman, J. Am. Chem. Soc., 1996, 118, 13071–13072;(b) D. H. Appella, L. A. Christianson, D. A. Klein, D. R. Powell,H. L. Huang, J. J. Barchi and S. H. Gellman, Nature, 1997, 387,381–384.
4 D. Seebach, S. Abele, K. Gademann, G. Guichard,T. Hintermann, B. Jaun, J. L. Mathews, J. V. Schreiber,L. Oberer, U. Hommel and H. Widmer, Helv. Chim. Acta,1998, 81, 932–982.
5 D. Seebach and T. Hintermann, Synlett, 1997, 437–438.6 (a) D. Seebach, S. Abele, K. Gademann and B. Jaun, Angew.
Chem., Int. Ed., 1999, 38, 1595–1597; (b) K. Gademann,T. Hintermann and D. Seebach, Curr. Med. Chem., 1999, 6,905–925.
7 (a) D. Seebach, T. Sifferlen, P. A. Mathieu, A. M. Hane,C. M. Krell, D. J. Bierbaum and S. Abele, Helv. Chim. Acta,2000, 83, 2849–2864; (b) D. Seebach, B. Jaun, R. Sebesta,R. I. Mathad, O. Flogel, M. Limbach, H. Sellner andS. Cottens, Helv. Chim. Acta, 2006, 89, 1801–1825.
8 K. Gademann, A. Hane, M. Rueping, B. Jaun and D. Seebach,Angew. Chem., Int. Ed., 2003, 43, 1534–1537.
9 I. A. Motorina, C. Huel, E. Quiniou, J. Mispelter, E. Adjadjand D. S. Grierson, J. Am. Chem. Soc., 2001, 123, 8–17.
10 (a) S. Krauthauser, L. A. Christianson, D. R. Powell andS. H. Gellman, J. Am. Chem. Soc., 1997, 119, 11719–11720;(b) Y. J. Chung, L. A. Christianson, H. E. Stanger, D. R.Powell and S. H. Gellman, J. Am. Chem. Soc., 1998, 120,10555–10556; (c) Y. J. Chung, B. R. Huck, L. A.Christianson, H. E. Stanger, S. Krauthauser, D. R. Powelland S. H. Gellman, J. Am. Chem. Soc., 2000, 122, 3995–4004.
11 (a) A. Hayen, M. A. Schmitt, N. Nagassa, K. A. Thomson andS. H. Gellman, Angew. Chem., Int. Ed., 2004, 43, 505–510;(b) S. De Pol, C. Zorn, C. D. Klein, O. Zerbe and O. Reiser,Angew. Chem., Int. Ed., 2004, 43, 511–514; (c) G. V. M.Sharma, P. Nagendar, P. Jayaprakash, P. R. Krishna,K. V. S. Ramakrishna and A. C. Kunwar, Angew. Chem., Int.Ed., 2005, 44, 5878–5882; (d) C. Baldauf, R. Gunther andH.-J. Hofmann, Biopolymers, 2006, 84, 408–413.
12 (a) W. S. Horne and S. H. Gellman, Acc. Chem. Res., 2008, 41,1399–1408; (b) L. K. A. Pilsl and O. Reiser, Amino Acids, 2011,41, 709–718.
13 (a) D. Balamurugan and K. M. Muraleedharan, Tetrahedron,2009, 65, 10074–10082; (b) D. Balamurugan and K. M.Muraleedharan, Chem. – Eur. J., 2012, 18, 9516–9520.
14 (a) G. Srinivasulu, S. K. Kumar, G. V. M. Sharma and A. C. Kunwar,J. Org. Chem., 2006, 71, 8395–8400; (b) G. V. M. Sharma,K. S. Reddy, S. J. Basha, K. R. Reddy and A. V. S. Sarma, Org.Biomol. Chem., 2011, 9, 8102–8111; (c) G. V. M. Sharma, T. Sridhar,P. Purushotham Reddy and A. C. Kunwar, Eur. J. Org. Chem., 2013,3543–3554; (d) G. V. M. Sharma, P. Nagendar, K. V. S.Ramakrishna, N. Chandramouli, M. Choudhary and A. C.Kunwar, Chem. – Asian J., 2008, 3, 969–983; (e) G. V. M. Sharma,N. Chandramouli, S. J. Basha, P. Nagendar, K. V. S. Ramakrishnaand A. V. S. Sarma, Chem. – Asian J., 2011, 6, 84–97; ( f ) G. V. M.Sharma, T. Prashanth, K. Sirisha, S. J. Basha, P. Gurava Reddy andA. V. S. Sarma, J. Org. Chem., 2014, 79, 8614–8628.
15 (a) G. V. M. Sharma, V. G. Reddy, A. S. Chander andK. R. Reddy, Tetrahedron: Asymmetry, 2002, 13, 21–24;(b) G. V. M. Sharma, K. Laxmi Reddy, P. Sree Lakshmi,R. Ravi and A. C. Kunwar, J. Org. Chem., 2006, 71,3967–3969; (c) D. Krishna, K. Laxmi Reddy and G. V. M.Sharma, Lett. Org. Chem., 2009, 6, 151–155.
16 (a) M. Lee, J. Shim, P. Kang, I. A. Guzei and S. H. Choi,Angew. Chem., Int. Ed., 2013, 52, 12564–12567; (b) W. Lee,S. Kwon, P. Kang, I. A. Guzei and S. H. Choi, Org. Biomol.Chem., 2014, 12, 2641–2644.
17 (a) B. Sundararaju, T. Sridhar, M. Achard, G. V. M. Sharmaand C. Bruneau, Eur. J. Org. Chem., 2010, 6092–6096;(b) B. Sundararaju, T. Sridhar, M. Achard, G. V. M. Sharmaand C. Bruneau, Eur. J. Org. Chem., 2013, 6433–6442.
18 S. Hanessian, H. Yang and R. Schaum, Synlett, 1996, 2507–2508.19 (a) L. C. Chan and G. B. Cox, J. Org. Chem., 2007, 72,
8863–8869; (b) M. Bodanszky, Peptide Chemistry: A PracticalTextbook, Springer, New York, 1988.
20 See the ESI†.21 Solvent titration studies were carried out by sequentially
adding up to 300 mL of DMSO-d6 in 600 mL of CDCl3 solutionof peptides.
22 (a) D. J. States, R. A. Haberkorn and D. J. Ruben, J. Magn.Reson., 1982, 48, 286–292; (b) D. Marion, M. Ikura,R. Tschudin and A. D. Bax, J. Magn. Reson., 1989, 85, 393–399.
23 Discover, Version 2.98, Biosym Molecular Simulations:SanDiego, CA, 1995.
NJC Paper
Publ
ishe
d on
23
Janu
ary
2015
. Dow
nloa
ded
by H
ebre
w U
nive
rsity
of
Jeru
sale
m o
n 03
/02/
2015
17:
53:4
1.
View Article Online
