Macrocyclization of amino acids or linear peptides upon reaction with amphoteric amino aldehydes and isocyanides is provided.
|
1. A cyclic compound of formula (III) e####
##STR00028##
wherein
n=0 or 1, and R1, R2, R3, R4 and R5 are independently selected from H; lower alkyl; aryl; heteroaryl; alkenyl; heterocycle; cyckoalkyl; esters of the formula C(O)OR* wherein R* is selected from alkyl and aryl; amides of the formula C(O)NR**R***, wherein R** and R*** are independently selected from alkyl and aryl; —CH2C(O)R, wherein R is selected from —OH, lower alkyl, aryl, -lower alkyl-aryl, or —NRaRb, where Ra and Rb are independently selected from H, lower alkyl, aryl or -lower alkyl-aryl; —C(O)Rc, wherein Rc is selected from lower alkyl, aryl or -lower alkyl-aryl; or -lower alkyl-ORd, wherein Rd is a suitable protecting group or oh group;
bonds [a] and [b] are syn or antito each other;
R′ is an amino acid side chain of the amino terminus amino acid;
R″ is an optionally substituted amide; and
R6 is a nucleophilic moiety of a compound having the formula R6—H, wherein R6—H is selected from the group consisting of 7-mercapto-4-methyl-coumarin, thiobenzoic acid, thioacetic acid, biotin, aromatic thiols, aliphatic thiols; and is capable of nucleophilic opening of the aziridine ring of a compound of formula (II)
##STR00029##
to form the compound of formula (III);
and the linear molecule is an amino acid, a linear peptide comprising 2-9 amino acids, or a salt of the amino acid or the linear peptide, wherein N′ is the nitrogen at the amino terminus end of the linear molecule and C′ is the carbon at the carboxy terminus end of the linear molecule, and provided that if the linear molecule is a linear peptide, bonds [a] and [c] are anti—to each other.
8. The cyclic compound of
9. The cyclic compound of
22. The cyclic compound of
23. The cyclic compound of
24. The cyclic compound of
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This application is a continuation of U.S. patent application Ser. No. 13/257,159, filed Nov. 21, 2011, pending, which is a U.S. National Stage filing of International Application Serial No. PCT/CA2010/000408, filed Mar. 16, 2010, which claims priority to U.S. Provisional Application Ser. No. 61/160,571, filed Mar. 16, 2009, each of which is incorporated herein by reference in their entirety.
The present invention relates to cyclic amino acid molecules and methods of preparing the same, and in particular the macrocyclization of amino acids or linear peptides upon reaction with amphoteric amino aldehydes and isocyanides.
Peptides play vital roles by mediating a wide range of biological processes, acting as hormones, antibiotics, and signaling molecules. Due to the highly specific interaction with their biological targets, peptides have been widely used in medicine. However, the enormous therapeutic potential of peptides is not always easy to realize due to their low bioavailability. This shortcoming is a consequence of the degradation of peptides by endo- and exopeptididases, which results in poor in vivo stability of peptides. Compared to their linear counterparts, cyclic peptides are more resistant to degradation. There are two main reasons for this stability. Firstly, exopeptidases cannot cleave the cyclic peptide at its (non-existent) ends. Secondly, cyclic peptides, especially those with a small-to-medium ring size, are protected against endopeptidases because the constrained cyclic peptide backbone prevents the adaptation of the required extended conformation during proteolysis. In addition, the reduced charge and intramolecular hydrogen bonding within cyclic peptides facilitate passive membrane permeability, which contributes to their enhanced bioavailability. Most significantly, conformational constraints imposed on the amino acid sequence by the cyclic topology maximize enthalpic interactions between cyclic peptides and their biochemical targets while ensuring favourable entropy of binding.
There has been enormous interest in both naturally occurring and synthetic cyclic peptides as scaffolds that pre-organize an amino acid sequence into a rigid conformation.1 Amongst the vast number of known cyclic peptides, rigid small-to-medium sized rings have been of particular interest. Various cyclolactamization and non-peptidic cyclization methods2 have been developed.
The macrocyclization of linear precursors is afflicted by several thermodynamic and kinetic challenges that arise from the conformational preferences of linear peptides. The chain/ring conformational equilibrium is the central obstacle facing synthesis of cyclic molecules from acyclic precursors. Short linear peptides can easily adopt a circular conformation, which is driven by ion pairing between the N- and C-termini (Scheme 1, A).3 Despite the unfavorable entropy, these circular conformations are thermodynamically favoured due to the enthalpy garnered through electrostatic and other polar interactions. As shown in Scheme 1, conventional activation reagents tend to remove the zwitterionic character of the peptide, rendering it incapable of forming ion pairs. Consequently, without enthalpic contribution from electrostatics and other polar interactions, the activated peptide adopts a random linear conformation (Scheme 1, B). In order for macrocyclization to occur, the activated peptide must adopt a pre-cyclization conformation (C) prior to forming the desired cyclic molecule (D). High dilution, on the order of 10−4 or greater, is essential to limiting the formation of by-products arising from cyclodimerization,4 cyclotrimerization, and polymerization.5 Unfortunately, dilution brings about long reaction times, which in turn provoke background processes such as epimerization. Amongst the most challenging cyclizations are those attempted on linear peptides containing less than seven residues.6,7
##STR00001##
Another common challenge in exploring macrocyclic chemistry space has to do with late-stage modification. This is a historic challenge for macrocyclic compound libraries built in the biotechnology and pharmaceutical companies. Their typical cyclization techniques (ring-closing metathesis, Huisgen cycloaddition) do not naturally lend themselves to further elaboration. Functional group handles must be built in prior to cyclization to achieve this goal.
In one aspect, there is provided a process to produce a cyclic amino acid molecule comprising reacting an amino acid molecule, having an amino terminus and a carboxyl terminus, with an isocyanide and a compound having formula (Ia) and/or (Ib):
##STR00002##
wherein:
In another aspect, there is provided a cyclic amino acid molecule prepared using the process described herein.
In another aspect, there is provided a cyclic amino acid molecule of formula (II):
##STR00003##
wherein,
In another aspect, there is provided use of a compound for cyclizing an amino acid molecule, wherein the compound has the formula (Ia)/(Ib):
##STR00004##
Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the description and drawings, like numerals refer to like structures or processes. In the drawings:
There is described herein a one-step process that provides cyclic peptides in high yields and selectivities while evading the problems typically encountered during traditional cyclization reactions. The late stage diversification of macrocyclic molecules can now be achieved in a seamless fashion. The products of the macrocyclization are equipped with specific modification sites, which enable late-stage structural modification of cyclic peptides. The post cyclization diversification has been a historic challenge for macrocycle libraries in both biotechnology and pharmaceutical industries. The present macrocyclization approach solves this problem, among others.
There is demonstrated herein a use for a class of stable amphoteric amino aldehydes that contain synthetically useful unprotected aziridine and aldehyde groups.8 Amphoteric amino aldehydes are coaxed into undergoing a reversible reaction with the amine and carboxyl ends of the peptide. The resulting electrophilic intermediate rapidly reacts with the nucleophilic aziridine portion due to the high effective molarity. As a result, the unfavorable chain/ring equilibrium is shifted using the Le Chatelier principle, securing efficient conversion to cyclic products. In another aspect, the cyclization tolerates aliphatic, acidic, and basic amino acid side chain residues. In particular, peptides, including those containing both acidic and basic residues, are found to be within the scope of this process, delivering a single diastereomeric product in each case. Trifluoroethanol (“TFE”), known for its capacity to stabilize peptide secondary structure and promote polar interactions,9 is the preferred reaction medium. The Ugi four-component condensation, a well-known reaction involving carboxylic acids, amines, aldehydes, and isocyanides10, is the preferred means of generating zwitterionic macrocyclization precursors. Mechanistically, the Ugi reaction is known to proceed through a series of reversible transformations that tend towards the thermodynamic driving force of amide bond formation. When an α-amino acid, aldehyde, and isocyanide are used as starting materials, the reaction traverses a zwitterionic iminium ion intermediate, which upon attack by the isocyanide produces an electrophilic mixed anhydride. Subsequent reaction with methanol gives a linear peptide ester as a mixture of diastereoisomers.11 This reaction has been attempted in the synthesis of cyclic peptides from linear peptides and conventional (monofunctional) aldehydes. However, low yields are accompanied by lack of diastereoselectivity and dominant cyclodimerization products.12,13
In one aspect, there is provided a process to produce a cyclic amino acid molecule comprising reacting an amino acid molecule, having an amino terminus and a carboxyl terminus, with an isocyanide and a compound having formula (Ia) and/or (Ib):
##STR00005##
wherein:
Preferably, if the amino acid molecule is an amino acid then the amino terminus is a primary amino group or a secondary amino group but wherein the amino acid molecule is a peptide, then the amino terminus is a secondary amino group.
In one embodiment, any one of R1-R5 is H. Preferably, n=0 and R2 and R3 is H or R1-R3 is H.
In a particular embodiment R1 is CH2OTBDMS or CH2iPr.
In one embodiment, the amino acid molecule is a linear peptide. Preferably, the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH2CH2SO2Ph or NHCH2CH2CN.
In another embodiment, the amino acid molecule is a D or L amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan and valine.
The amino acid molecule may be an alpha-amino acid, beta-amino acid or gamma-amino acid.
In one embodiment the isocyanide is selected from the group consisting of: (S)-(−)-α-Methylbenzyl isocyanide; 1,1,3,3-Tetramethylbutyl isocyanide; 1-Pentyl isocyanide; 2,6-Dimethylphenyl isocyanide; 2-Morpholinoethyl isocyanide; 2-Naphthyl isocyanide; 2-Pentyl isocyanide; 4-Methoxyphenyl isocyanide; Benzyl isocyanide; Cutyl isocyanide; Cyclohexyl isocyanide; Isopropyl isocyanide; p-Toluenesulfonylmethyl isocyanide; Phenyl isocyanide dichloride; tert-Butyl isocyanide; (Trimethylsilyl)methyl isocyanide; 1H-Benzotriazol-1-ylmethyl isocyanide; 2-Chloro-6-methylphenyl isocyanide; Di-tert-butyl 2-isocyanosuccinate; tert-Butyl 2-isocyano-3-methylbutyrate; tert-Butyl 2-isocyano-3-phenylpropionate; tert-Butyl 2-isocyanopropionate; and tert-Butyl 3-isocyanopropionate, preferably, tert-Butyl isocyanide.
In some embodiments, the process is conducted in a non-nucleophilic reaction medium, preferably trifluoroethanol or HFIP mixed with water.
In one embodiment, if the amino acid molecule is an amino acid, the process is conducted in water.
In some embodiments, the process further comprises conjugating a fluorescent tag to the cyclic amino acid molecule by nucleophilic ring-opening of the aziridine moiety.
In some embodiments, the peptide is between 2 and 30 amino acids in length.
In another aspect, there is provided a cyclic amino acid molecule prepared using the process described herein.
One would understand that cyclization, in some cases, may require and would include protecting certain peptide or amino acid side chains in manner known to a person skilled in the art.
In another aspect, there is provided a cyclic amino acid molecule of formula (II):
##STR00006##
wherein,
Preferably, if the amino acid molecule is an amino acid then the amino terminus is a primary amino group or a secondary amino group but wherein the amino acid molecule is a linear peptide, then the amino terminus is a secondary amino group.
In one embodiment, any one of R1-R5 is H. Preferably, n=0 and R2 and R3 is H or R1-R3 is H.
In a particular embodiment R1 is CH2OTBDMS or CH2iPr.
In one embodiment, the amino acid molecule is a linear peptide. Preferably, the amino terminus amino acid of the linear peptide is selected from the group consisting of proline and an amino acid with an amino group substituted with NHBn, NHCH2CH2SO2Ph or NHCH2CH2CN.
In another embodiment, the amino acid molecule is a D or L amino acid selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan and valine.
The amino acid molecule may be an alpha-amino acid, beta-amino acid or gamma-amino acid.
In one embodiment R″ is tert-Butyl amide.
In another aspect, there is provided use of a compound for cyclizing an amino acid molecule, wherein the compound has the formula (Ia)/(Ib):
##STR00007##
As used herein, the term “amino acid molecule” is meant to include single amino acids and also peptides.
As used herein, the term “amino acid” refers to molecules containing an amine group, a carboxylic acid group and a side chain that varies. Amino acid is meant to include not only the twenty amino acids commonly found in proteins but also non-standard amino acids and unnatural amino acid derivatives known to those of skill in the art, and therefore includes, but is not limited to, alpha, beta and gamma amino acids. Peptides are polymers of at least two amino acids and may include standard, non-standard, and unnatural amino acids.
The term “suitable substituent” as used in the context of the present invention is meant to include independently H; hydroxyl; cyano; alkyl, such as lower alkyl, such as methyl, ethyl, propyl, n-butyl, t-butyl, hexyl and the like; alkoxy, such as lower alkoxy such as methoxy, ethoxy, and the like; aryloxy, such as phenoxy and the like; vinyl; alkenyl, such as hexenyl and the like; alkynyl; formyl; haloalkyl, such as lower haloalkyl which includes CF3, CCl3 and the like; halide; aryl, such as phenyl and napthyl; heteroaryl, such as thienyl and furanyl and the like; amide such as C(O)NRaRb, where Ra and Rb are independently selected from lower alkyl, aryl or benzyl, and the like; acyl, such as C(O)—C6H5, and the like; ester such as —C(O)OCH3 the like; ethers and thioethers, such as O-Bn and the like; thioalkoxy; phosphino; and NRaRb, where Ra and Rb are independently selected from lower alkyl, aryl or benzyl, and the like. It is to be understood that a suitable substituent as used in the context of the present invention is meant to denote a substituent that does not interfere with the formation of the desired product by the processes of the present invention.
As used in the context of the present invention, the term “lower alkyl” as used herein either alone or in combination with another substituent means acyclic, straight or branched chain alkyl substituent containing from one to six carbons and includes for example, methyl, ethyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, and the like. A similar use of the term is to be understood for “lower alkoxy”, “lower thioalkyl”, “lower alkenyl” and the like in respect of the number of carbon atoms. For example, “lower alkoxy” as used herein includes methoxy, ethoxy, t-butoxy.
The term “alkyl” encompasses lower alkyl, and also includes alkyl groups having more than six carbon atoms, such as, for example, acyclic, straight or branched chain alkyl substituents having seven to ten carbon atoms.
The term “aryl” as used herein, either alone or in combination with another substituent, means an aromatic monocyclic system or an aromatic polycyclic system. For example, the term “aryl” includes a phenyl or a napthyl ring, and may also include larger aromatic polycyclic systems, such as fluorescent (eg. anthracene) or radioactive labels and their derivatives.
The term “heteroaryl” as used herein, either alone or in combination with another substituent means a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur and which form an aromatic system. The term “heteroaryl” also includes a polycyclic aromatic system comprising a 5, 6, or 7-membered unsaturated heterocycle containing from one to 4 heteroatoms selected from nitrogen, oxygen, and sulphur.
The term “cycloalkyl” as used herein, either alone or in combination with another substituent, means a cycloalkyl substituent that includes for example, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
The term “cycloalkyl-alkyl-” as used herein means an alkyl radical to which a cycloalkyl radical is directly linked; and includes, but is not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl and 2-cyclohexylethyl. A similar use of the “alkyl” or “lower alkyl” terms is to be understood for aryl-alkyl-, aryl-loweralkyl- (eg. benzyl), -lower alkyl-alkenyl (eg. allyl), heteroaryl-alkyl-, and the like as used herein. For example, the term “aryl-alkyl-” means an alkyl radical, to which an aryl is bonded. Examples of aryl-alkyl- include, but are not limited to, benzyl (phenylmethyl), 1-phenylethyl, 2-phenylethyl and phenylpropyl.
As used herein, the term “heterocycle”, either alone or in combination with another radical, means a monovalent radical derived by removal of a hydrogen from a three- to seven-membered saturated or unsaturated (including aromatic) heterocycle containing from one to four heteroatoms selected from nitrogen, oxygen and sulfur. Examples of such heterocycles include, but are not limited to, aziridine, epoxide, azetidine, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, hydantoin, diazepine, imidazole, isoxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine, homo-piperazine, 1,4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine, pyridine-N-oxide or pyrimidine, and the like.
The term “alkenyl”, as used herein, either alone or in combination with another radical, is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a double bond. Examples of such radicals include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl.
The term “alkynyl”, as used herein is intended to mean an unsaturated, acyclic straight chain radical containing two or more carbon atoms, at least two of which are bonded to each other by a triple bond. Examples of such radicals include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.
The term “alkoxy” as used herein, either alone or in combination with another radical, means the radical —O—(C1-n)alkyl wherein alkyl is as defined above containing 1 or more carbon atoms, and includes for example methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1,1-dimethylethoxy. Where n is 1 to 6, the term “lower alkoxy” applies, as noted above, whereas the term “alkoxy” encompasses “lower alkoxy” as well as alkoxy groups where n is greater than 6 (for example, n=7 to 10). The term “aryloxy” as used herein alone or in combination with another radical means —O-aryl, wherein aryl is defined as noted above.
A peptide is a polymer of two or more amino acids.
The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
Anhydrous toluene and dimethylformamide (DMF) were purchased and used as received. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl under argon. All other solvents including TFE (2,2,2-trifluoroethanol) and HFIP (1,1,1,3,3,3-hexafluoro-2-isopropanol) were of reagent grade quality. Melting points were obtained on a MelTemp melting-point apparatus and are uncorrected.
Method 1: amino acid/peptide cyclization reaction was conducted using the following method. In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of TFE and stirred until a homogeneous solution has been obtained. Aziridine aldehyde dimer (0.1 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in Table 1. Reactions were monitored by electrospray ionization mass spectrometry (“ESI-MS”) at 60 eV and/or thin layer chromatography (“TLC”) analysis. After completion of the reaction, 1 ml of water and 1 ml of Et2O were added and the mixture was shaken vigorously and then cooled on ice. The resulting precipitate was filtered and washed with hexanes and cold Et2O (1 ml) to afford the cyclic peptide. For products that are water soluble or do not precipitate, the reaction mixture was concentrated under reduced pressure and then triturated with Et2O and hexanes (0.2 ml) to afford the cyclic peptide product.
Method 2: amino acid/peptide cyclization reaction was conducted using HFIP in place of TFE (Method 2). In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of HFIP and stirred until homogeneous solution has been obtained. Aziridine aldehyde dimer (0.1 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in Table 1. Reactions were monitored by ESI-MS at 60 eV and/or TLC analysis. After completion of the reaction, the mixture was concentrated under reduced pressure and then triturated with Et2O and hexanes (0.2 ml) to afford the cyclic peptide product.
Method 3: amino acid/peptide cyclization reaction was carried out using HFIP and water. In a screw-cap vial equipped with a magnetic stirring bar was added peptide (0.2 mmol) and 1 ml of HFIP and 47 microliters of H2O and stirred until homogeneous. Aziridine aldehyde dimer (0.1 mmol) and isocyanide (0.2 mmol) were then added sequentially and the resulting mixture was stirred for the time specified in Table 1. Reactions were monitored by ESI-MS at 60 eV and/or TLC analysis. After completion of the reaction, the mixture was concentrated under reduced pressure and then triturated with Et2O and hexanes to afford the cyclic peptide product.
Note: In reactions involving either cysteine or peptides containing thiol residues, the solvent was degassed with argon for two hours prior to reaction. The reaction was thereafter carried out under an atmosphere of argon.
Some of the cyclic products that may be synthesized from each of Methods 1-3 are summarized in Table 3 and depicted in
TABLE 3
Methods used for amino acid/peptide cyclization reactions.
Method
Cyclic Products
1
1, 2, 3, 4, 7, 9, 11, 12, 15, 16, 17, 18,
19, 20, 21, 22
2
5, 8, 10, 13, 14
3
6
The relative stereochemistry of each cyclic product listed in Table 3 was established by correlating the methine region of each 1H NMR spectrum with that of piperazinone 1.
Chromatography: Flash column chromatography was carried out using Silicycle 230-400 mesh silica gel. Thin-layer chromatography (TLC) was performed on Macherey Nagel pre-coated glass backed TLC plates (SIL G/UV254, 0.25 mm) and visualized using a UV lamp (254 nm) and iodine stain.
Nuclear magnetic resonance spectra: 1H and 13C NMR spectra were recorded on Varian Mercury 400 or 500 MHz spectrometers. 1H NMR spectra were referenced to TMS (0 ppm) and 13C NMR spectra were referenced to CDCl3 (77.23 ppm). Peak multiplicities are designated by the following abbreviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; ds, doublet of singlets; dd, doublet of doublets; ddd, doublet of doublet of doublets; bt, broad triplet; td, triplet of doublets; tdd, triplet of doublets of doublets.
Mass Spectroscopy: High-resolution mass spectra were obtained on a VG 70-250S (double focusing) mass spectrometer at 70 eV or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and accurate mass capabilities. Low resolution mass spectra (ESI) were obtained at 60 eV, 70 eV and 100 eV.
Synthetic routes toward aziridine aldehyde dimers: All dimeric amino aldehydes used in this study were prepared according to the synthetic route outlined in Supplementary
Results
Diverse piperazinones were produced in a one-step process (Table 1).
TABLE 1
The scope of piperazinone synthesis
(L-amino acid-derived product is shown).
##STR00008##
##STR00009##
##STR00010##
Entrya
R1
Amino Acid
Time
Productb
1
CH2OTBDMS
L-Phe
1
1 (92)
2
CH2OTBDMS
D-Phe
1.5
2 (90)
3
CH2OTBDMS
L-Ala
1.5
3 (82)
4
CH2OTBDMS
L-Pro
1
4 (98)
5
CH2iPr
D-Arg
1
5 (83)
6c
CH2iPr
L-Lys HClg
1
6 (76)
7
CH2OTBDMS
L-Gly
1
7 (80)
8e
CH2iPr
L-Asp
3
8 (76)
9f
CH2OTBDMS
L-Cys
2
9 (82)
10d
CH2OTBDMS
L-His
1
10 (88)
11
CH2OTBDMS
L-Ser
3
11 (77)
aUnless specified otherwise, reactions were performed at room temperature using 0.2 mmol
of isocyanide and amino acid, and 0.1 mmol of amino aldehyde dimer in TFE (0.2 M).
b% isolated yield.
cHFIP/H2O (20:1) was used as solvent (0.2 M).
dHFIP was used as solvent (0.2 M).
eCyclization occurs at the more acidic α-carboxylic acid.
fTFE was degassed prior to use.
gLysine side chain amine is protonated.
The step that shifts the ring/chain equilibrium is transannular collapse of the nucleophilic NH aziridine onto the electrophilic mixed anhydride generated when an amphoteric amino aldehyde is subjected to the Ugi reaction condition (Scheme 2). The reaction takes place under stoichiometric conditions and delivers cyclic peptide product. High dilution, a critical condition normally required in order to achieve high yields in cyclic peptide synthesis, is not necessary.
##STR00011##
The normally challenging medium-sized rings can be made with great facility and in good yields within several hours (Table 2).
TABLE 2
Representative scope of linear peptide macrocyclization.
##STR00012##
##STR00013##
##STR00014##
##STR00015##
12
##STR00016##
13
##STR00017##
14
##STR00018##
15
##STR00019##
16
##STR00020##
17
##STR00021##
18
##STR00022##
19
##STR00023##
20
##STR00024##
21
##STR00025##
22
aUnless specified otherwise, reactions were performed at room temperature using 0.2 mmol
of isocyanide and amino acid, and 0.1 mmol of amino aldehyde dimer in TFE (0.2 M).
bIsolated yield.
cDiastereoselectivity of >20:1 was confirmed by 1H NMR analysis of each crude reaction
mixture. The relative stereochemistry was established by correlating the methine region of
each 1H NMR spectrum with that of piperazinone 1 (see X-ray crystal structure of 1).
dHFIP was used as solvent (0.2 M).
1H and 13C NMR spectra for the cyclic products of Table 3 are shown in
A one-step process for making versatile cyclic peptides with high stereo- and chemoselectivity for linear peptides, isocyanides, and amphoteric amino aldehydes is provided. The Ugi reaction is run with an amphoteric amino aldehyde in place of a monofunctional aldehyde. When L-phenyl alanine was reacted with tert-butyl isocyanide, and an aziridine aldehyde, a cyclic piperazinone was obtained in 92% yield in approximately one hour (Table 1, Entry 1). The relative stereochemistry of 1 was established using X-ray analysis. A range of amino acids were subjected to this reaction and in all cases the corresponding piperazinone products were obtained as single diastereoisomers without formation of linear peptides (
The existence of epimeric products were investigated through the use of 1H NMR (DMSO-d6 or CDCl3) analysis of crude reaction mixtures. In all of the reactions studied, there were no detectable signals corresponding to the epimer. As such, the resulting crude cyclic peptides were obtained in >20:1 selectivity.
The effect of medium-sized peptide chain length on the reaction outcome was studied (Table 2). The challenging medium-sized rings are readily prepared; the reaction times are less than 10 hours, proceeding with high yields and diastereoselectivities. The preferred work-up procedure involves product precipitation from diethyl ether and hexanes. The cyclic peptides require no further purification by HPLC. In addition, racemization was not detected throughout the course of the reaction or during product isolation. The lack of epimerization is further evidenced by high stereoselectivity; aziridine aldehydes with S stereocenters next to the carbonyl group undergo macrocyclization with the peptides that contain an L-amino acid residue at the N-terminus. The “mismatched” reaction with the D-amino acid-terminated peptide is unproductive, leading to the formation of stable aminals.
It has been determined that the α-stereocenter of the N-terminal amino acid, in this case proline, should match the α-stereocenter of the aziridine aldehyde in order for cyclization to occur, with respect to peptides. In a mismatched reaction, the only product formed is the corresponding aminal (
When the opposite enantiomer of amino aldehyde was used in the same reaction, the proline and the amino aldehyde both had S-configuration at their α-stereocenters. This matched case readily underwent peptide cyclization to yield the corresponding cyclic product in high yield (
This stereoselectivity process is significant in the context of possible epimerization at the N-terminus. Since matched stereochemistry is required for the cyclization to occur, the resulting cyclic peptides will necessarily contain matching stereocenters. Furthermore, the fact that no cyclic peptide formation was observed in mismatched reactions suggests that no epimerization, which would result in matched substrates, occurs under our macro conditions.
Consistent yields in this chemistry are likely obtained due to the mechanism that governs cyclization. It is at this point that a departure from the Ugi reaction with monofunctional aldehydes becomes apparent. When monofunctional aldehydes are used in the reaction with isocyanides and peptides, low diastereoselectivities are observed. More importantly, the undesired cyclodimerization occurs during the cyclization of linear peptides containing less than six residues; the cyclization of tripeptides yields only the cyclodimers.13 This low selectivity is due to a slow transannular attack of the amine onto the mixed anhydride (Scheme 2, A), thus allowing the intermolecular process to be kinetically competitive. By adding an amphoteric amino aldehyde to the cyclization reaction mixture containing a secondary amine-terminated peptide, the slow transannular attack is being replaced with a fast attack by the nucleophilic aziridine, which is positioned exocyclic to the mixed anhydride (Scheme 2, B). This provides an unencumbered trajectory of attack. Since TFE is a non-nucleophilic solvent, premature solvolysis of the mixed anhydride is not observed. The reaction mechanism ensures that the C-terminus is activated only upon formation of the intermediate cyclic mixed anhydride, which is then attacked by the exocyclic aziridine. This not only secures selectivity for the intramolecular macrocyclization, but also avoids prolonged C-terminus activation and potential epimerization. Accordingly, high dilution, a critical condition normally required in order to achieve high yields in conventional cyclic peptide synthesis, is not necessary.6 Furthermore, no oligomeric or polymeric by-products were detected in the subject experiments. A side-by side comparison of the subject reaction with the traditional lactamization of a linear tetrapeptide, widely used in the synthesis of cyclic peptides, was conducted. The amino aldehyde mediated macrocyclization delivered rapid, selective, and efficient formation of the cyclic peptide. In contrast, only trace amount of the cyclic peptide was detected among myriad structures formed in the course of the lactamization. The undesired cyclodimerization5 has dominated this process, which is a testament to sluggish kinetics of the conventional intramolecular process. Peptide macrocyclization reactions are normally performed under extremely dilution. Typical protocols necessitate molar concentrations of 10−4 M. This large dilution increases the selectivity for intramolecular cyclizations, thereby limiting undesired cyclodimerization and trimerization.
Characterization of the relative stereochemistry of each cyclic product listed in Table 3 is set out below.
1H NMR (CDCl3, 400 MHz) δ: 7.36-7.18 (m, 5H), 6.75 (bs, 1H), 3.86 (dd, J=11.6 Hz, 4.8 Hz, 1H), 3.74 (dd, J=11.2 Hz, 4.8 Hz, 1H), 3.54 (bs, 1H), 3.47 (dd, J=3.6 Hz, 1.2 Hz, 1H), 3.25 (dd, J=14.4 Hz, 3.2 Hz, 1H), 3.10 (m, 1H), 2.52 (dd, J=14.4 Hz, 10.4 Hz, 1H), 2.40 (m, 1H), 1.80 (bs, 1H), 1.02 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz) δ: 184.8, 169.0, 138.5, 129.7, 128.9, 127.0, 63.9, 57.5, 53.6, 50.7, 44.6, 39.0, 35.5, 28.5, 26.1, 18.6, −5.0 ppm. HRMS (ESI) [MH]+ calcd. 446.2833. found 446.2831.
1H NMR (CDCl3, 400 MHz) δ: 7.35-7.20 (m, 5H), 6.32 (bs, 1H), 3.86 (m, 1H), 3.73 (dd, J=11.5 Hz, 4.2 Hz, 1H), 3.86 (bs, 1H), 3.73 (dd, J=11.5 Hz, 4.2 Hz, 1H), 3.25 (dd, J=14.4 Hz, 3.2 Hz, 1H), 3.10 (m, 1H), 2.52 (dd, J=14.4 Hz, 10.4 Hz, 1H), 2.40 (m, 1H), 1.80 (bs, 1H), 1.31 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. 13C NMR (CDCl3, 75 MHz) δ: 184.0, 169.1, 136.2, 129.2, 129.1, 127.3, 63.0, 59.5, 51.4, 49.6, 42.2, 38.7, 36.0, 28.8, 26.1, 18.7, −5.1 ppm. MS (ESI) [MH]+ calcd. 446.3. found 446.3.
1H NMR (CDCl3, 400 MHz) δ: 7.42 (s, NH), 3.88 (dd, J=11.2 Hz, 4.8 Hz, 1H), 3.70 (dd, J=11.2 Hz, 5.2 Hz, 1H), 3.55 (d, J=0.8 Hz, 1H), 3.46 (dd, J=3.6 Hz, 1.6 Hz, 1H), 3.07 (m, 1H), 2.36 (td, J=4.8 Hz, 3.6 Hz, 1H), 1.8 (bs, NH), 1.39 (s, 9H), 1.23 (d, J=6.8 Hz, 3H), 0.90 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz) δ: 185.5, 169.3, 63.9, 53.8, 51.2, 51.0, 44.5, 39.3, 28.9, 26.1, 16.6, 15.0, −5.0 ppm. HRMS (ESI) [MH]+ calcd. 370.2520. found 370.2520.
1H NMR (CDCl3, 400 MHz) δ: 6.17 (s, 1H), 3.76 (dd, J=11.6 Hz, 4.4 Hz, 1H), 3.67 (dd, J=11.6 Hz, 4.8 Hz, 1H), 3.70 (d, J=6.2 Hz, 1H), 3.12 (dt, J=4.8 Hz, 9.2 Hz, 1H), 2.97 (dd, J=6.4 Hz, 3.6 Hz, 1H), 2.93-2.89 (m, 1H), 2.60 (q, J=4.4 Hz, 1H), 2.25-2.05 (m, 2H), 1.90-1.75 (m, 3H), 1.70-1.51 (m, 1H), 1.48 (s, 9H), 0.85 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz) δ: 183.0, 168.6, 65.0, 63.4, 63.2, 54.6, 51.3, 43.4, 41.0, 29.0, 26.1, 22.2, 22.0, 18.9, −5.1 ppm. HRMS (ESI) [MH]+ calcd. 396.2676. found 396.2656.
1H NMR (D2O, 400 MHz) δ: 3.59 (t, J=5.6 Hz, 1H), 3.46 (m, 1H), 3.13 (m, 1H), 2.94 (m, 1H), 3.37 (m, 1H), 1.78-1.44 (m, 8H), 1.22 (s, 9H), 0.83 (d, J=2.8 Hz, 3H), 0.81 (d, J=2.8 Hz, 3H) ppm. 13H NMR (D2O, 400 MHz) δ: 186.7, 172.0, 156.9, 54.9, 54.5, 54.0, 44.5, 42.8, 28.7, 27.9, 26.5, 25.7, 25.0, 24.1, 22.2, 21.5 ppm. MS (ESI) [MH]+ calcd. 367.3. found 367.2. and 184.1 for [M+2H]2+/2.
NMR (D2O with 10% v/v CD3COOD, 400 MHz) δ: 5.14 (ddd, J=9.6 Hz, 4.4 Hz, 2.4 Hz, 1H), 4.12 (d, J=2.4 Hz, 1H), 3.07 (dd, J=7.6 Hz, 4.4 Hz, 1H), 2.85 (t, J=7.6 Hz, 2H), 1.82-1.46 (m, 10H), 1.32 (s, 9H), 0.83 (d, J=6.8 Hz, 3H), 0.81 (d, J=6.8 Hz, 3H) ppm. 13C NMR (D2O with 10% v/v CD3COOD, 100 MHz) δ: 178.0, 173.8, 97.2, 58.6, 54.5, 53.0, 40.6, 39.3, 29.3, 27.8, 27.6, 27.2, 26.8, 24.6, 22.2, 21.9, 20.8 ppm. MS (ESI) calcd. 339.3. found 339.2 and 170.1 for [M+2H]2+/2.
1H NMR (400 MHz, CDCl3) δ: 6.98 (s, 1H), 3.87 (dd, J=11.5, 4.5 Hz, 1H), 3.78 (dd, J=11.5, 4.5 Hz, 1H), 3.34-3.24 (m, 2H), 3.13 (d, J=4.6 Hz, 1H), 2.40 (td, J=4.5, 3.4 Hz, 1H), 2.17-1.96 (m, 1H), 1.43-1.36 (m, 9H), 0.94-0.86 (m, 9H), 0.13-0.05 (m, 6H). 13C NMR (101 MHz, CDCl3) δ: 183.93, 168.37, 77.48, 77.16, 76.84, 63.02, 55.81, 51.25, 48.72, 45.27, 39.64, 28.87, 26.06, 18.55, 0.15, −5.09, −5.12.
1H NMR (400 MHz, CD3OD) δ: 4.69-4.58 (m, 1H), 3.92 (d, J=2.0 Hz, 1H), 3.76 (dd, J=9.8, 3.6 Hz, 1H), 3.66 (dd, J=7.8, 4.0 Hz, 1H), 2.90 (dd, J=17.5, 3.6 Hz, 1H), 2.81 (dd, J=16.7, 4.0 Hz, 1H), 2.69-2.53 (m, 2H), 1.36-1.32 (m, 9H), 0.98 (dt, J=8.5, 4.2 Hz, 6H). 13C NMR (101 MHz, CD3OD) δ 188.21, 172.40, 158.66, 158.62, 56.05, 55.25, 55.23, 51.95, 44.17, 43.55, 43.35, 42.55, 42.39, 42.16, 41.98, 29.85, 28.90, 28.85, 28.10, 27.37, 27.16, 26.85, 26.51, 23.19, 22.63, 21.05.
Note: proper spectroscopic characterization of this compound is difficult as spectra are characterized by extremely broad peaks. This is attributed to inherent instability of 9 due to the presence of nucleophilic thiol and its susceptibility to oxidation and aziridine ring-opening.
1H NMR (CDCl3, 400 MHz) δ: 3.47 (s, 1H), 3.41 (d, J=6.5 Hz, 1H), 4.35-2.42 (m, 7H), 1.70-1.42 (bs, 10H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. MS (ESI) [MH]+ calcd. 402.6. found 402.2.
1H NMR (CDCl3, 400 MHz) δ: 7.56 (s, 1H), 7.25 (s, 1H), 6.87 (s, 1H), 6.22 (s, 1H), 4.46 (m, 1H), 3.72 (dd, J=11.2 Hz, 4.8 Hz, 1H), 3.60 (m, 1H), 3.44 (dd, J=3.6 Hz, 1.5 Hz, 1H), 3.27 (dd, J=14.1 Hz, 3.1 Hz, 1H), 2.99 (dd, J=14.7 Hz, 4.2 Hz, 1H), 2.80 (dd, J=15.2 Hz, 8.1 Hz, 1H), 2.40 (m, 1H), 2.05 (bs, 1H), 1.36 (s, 9H), 0.87 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz) δ: 185.4, 171.4, 169.5, 168.6, 135.1, 64.3, 62.7, 60.6, 54.7, 51.2, 44.5, 39.3, 28.9, 26.0, 18.4, −5.3 ppm. MS (ESI) [MH]+ calcd. 436.6. found 436.2.
1H NMR (CDCl3, 400 MHz) δ: 6.80 (bs, 1H), 6.15 (bs, 1H), 4.02 (d, J=11.6 Hz, 1H), 3.85 (dd, J=13.3 Hz, 6.7 Hz, 1H), 3.76 (dd, J=14.2 Hz, 3.0 Hz, 1H), 3.68 (bs, 1H), 3.53 (d, J=5.0 Hz, 1H), 3.45 (dd, J=3.5 Hz, 2.4 Hz, 1H), 3.13 (m, 1H), 2.55 (m, 1H), 2.42 (m, 1H), 1.39 (s, 9H), 0.90 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz) δ: 185.3, 168.9, 63.5, 61.4, 55.8, 53.9, 51.2, 44.9, 39.6, 28.9, 26.1, 18.6, −5.1 ppm. MS (ESI) [MH]+ calcd. 386.6. found 386.2.
1H NMR (CD3OD, 400 MHz) δ: 4.47 (m, 1H), 4.14 (dd, J=8.4 Hz, 6 Hz, 1H), 4.07-3.99 (m, 2H), 3.84 (dd, J=12 Hz, 3.2 Hz, 1H), 3.32 (d, J=5.6 Hz, 1H), 3.26-3.15 (m, 2H), 2.42-2.30 (m, 2H), 1.92-1.78 (m, 3H), 1.74 (q, J=6.8 Hz, 2H), 1.59 (sept, J=6.8 Hz, 1H), 1.22 (s, 9H), 0.90 (d, J=6.8 Hz, 3H), 0.87 (d, J=6.4 Hz, 3H), 0.84 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H) ppm. 13C NMR (CD3OD, 100 MHz) δ: 174.1, 164.1, 157.4, 86.5, 62.4, 60.7, 60.6, 57.7, 54.2, 52.1, 50.2, 39.6, 29.0, 25.8, 25.2, 25.1, 22.1, 21.0, 20.5, 17.9, −6.5 ppm. MS (ESI) calcd. 509.3. found 509.3.
1H NMR (CD3OD, 400 MHz) δ: 4.26 (t, J=5.6 Hz, 1H), 4.18 (dd, J=8.8 Hz, 2.4 Hz, 1H), 3.58 (dd, J=8.8 Hz, 5.2 Hz, 1H), 3.28-3.24 (m, 2H), 3.00 (d, J=5.6 Hz, 1H), 2.70 (dd, J=10.4 Hz, 6.4 Hz, 1H), 2.32-2.18 (m, 2H), 1.98-1.52 (m, 11H), 1.28 (s, 9H), 1.01 (d, J=6.4 Hz, 3H), 0.96 (d, J=6.4 Hz, 3H) ppm. 13C NMR (CD3OD, 100 MHz) δ: 178.0, 160.8, 159.1, 157.4, 86.9, 64.3, 63.5, 60.2, 54.5, 53.7, 51.0, 42.6, 41.4, 29.4, 28.8, 26.1, 24.9, 24.5, 22.7, 21.3, 20.4 ppm. MS(ESI) [MH]+ calcd. 464.3. found 464.3 and 232.6 for [M+2H]2+/2.
1H NMR (CD3OD, 400 MHz) δ: 4.46 (t, J=9.2 Hz, 1H), 4.34 (dd, J=8.4 Hz, 2.0 Hz, 1H), 3.81 (dd, J=8.4 Hz, 5.6 Hz, 1H), 3.38-3.30 (m, 1H), 3.02 (dd, J=17.2 Hz, 2.4 Hz, 1H), 2.85-2.73 (m, 1H), 2.57 (dd, J=17.2 Hz, 8.4 Hz, 1H), 2.45-2.32 (m, 2H), 2.05-1.62 (m, 7H), 1.58-1.48 (m, 2H), 1.27 (s, 9H), 1.01 (d, J=6.4 Hz, 3H), 0.98 (d, J=6.4 Hz, 3H) ppm. MS (ESI) [MH]+ calcd. 423.3. found 423.2.
1H NMR (CD3OD, 400 MHz) δ: 4.55 (ddd, J=8 Hz, 3.2 Hz, 1.6 Hz, 1H), 4.32 (dd, J=10.4 Hz, 4 Hz, 1H), 4.21 (dd, J=14 Hz, 6 Hz, 1H), 4.15-4.05 (m, 3H), 3.91 (dd, J=12 Hz, 3.2 Hz, 1H), 3.41 Hz (dd, J=6 Hz, 1H), 3.36-3.28 (m, 1H), 2.50-2.30 (m, 2H), 2.00-1.50 (m, 7H), 1.31 (s, 9H), 0.98-0.92 (m, 6H), 0.92 (s, 9H), 0.13 (s, 3H), 0.11 (s, 3H) ppm. 13C NMR (CD3OD, 100 MHz) δ: 178.3, 166.4, 165.1, 157.6, 86.7, 62.7, 61.0, 60.1, 54.2, 53.7, 52.6, 50.5, 44.0, 41.6, 28.9, 25.6, 25.2, 25.1, 25.1, 22.6, 20.7, 20.5, 17.9, −6.5, −6.5 ppm. MS (ESI) [MH]+ calcd. 566.4. found 566.4.
1H NMR (CDCl3, 500 MHz) δ: 7.82 (bs, NH), 7.40-7.20 (m, 5H), 7.18 (bs, NH), 7.05 (bs, NH), 6.81 (bs, 2 NH), 4.64 (q, J=6.8 Hz, 1H), 4.13 (dd, J=16 Hz, 7.6 Hz, 1H), 4.10-3.90 (m, 2H), 3.57 (dd, J=16 Hz, 4.8 Hz, 1H), 3.50-3.39 (m, 2H), 3.26 (dd, J=14 Hz, 5.6 Hz, 1H), 3.12 (dd, J=14 Hz, 7.2 Hz, 1H), 3.12-3.08 (m, 1H), 3.00-2.92 (m, 2H), 2.63 (t, J=3.6 Hz, 1H), 2.28-2.18 (m, 1H), 1.90-1.70 (m, 2H), 1.37-1.25 (m, 9H), 1.10-0.88 (m, 12H) ppm. 13C NMR (CDCl3, 125 MHz) δ: 181.4, 176.1, 175.7, 172.1, 170.1, 169.2, 136.4, 130.1, 128.7, 126.8, 56.2, 63.0, 43.2, 42.5, 40.2, 38.6, 37.9, 31.2, 30.0, 29.8, 29.5, 28.8, 28.7, 27.2, 25.1, 24.9, 24.3, 23.3, 23.1, 22.8, 22.7, 22.6, 22.4, 22.3, 21.9, MS (ESI) [MH]+ calcd. 682.4. found 682.4.
1H NMR (400 MHz, CDCl3) δ: 4.57-4.44 (m, 1H), 4.32-3.70 (m, 10H), 3.38 (d, J=5.4 Hz, 1H), 3.28 (s, 1H), 3.14 (dd, J=11.2, 5.6 Hz, 1H), 2.52-2.41 (m, 1H), 2.37 (d, J=6.9 Hz, 1H), 2.15-1.65 (m, 8H), 1.64-1.48 (m, 2H), 1.42-1.23 (m, 8H), 1.00 (dt, J=21.4, 7.4 Hz, 6H).
13C NMR (100 MHz, CD3OD) δ: 176.18, 171.23, 168.97, 165.90, 159.24, 86.37, 65.77, 63.49, 61.48, 59.76, 55.41, 51.43, 47.38, 45.65, 45.23, 44.59, 44.43, 44.19, 43.41, 42.46, 29.85, 29.43, 28.97, 28.58, 28.46, 27.72, 27.13, 26.74, 25.68, 23.84, 23.68, 23.14, 23.06, 22.16, 21.62.
1H NMR (400 MHz, CDCl3) δ: 7.84 (d, J=9.7 Hz, 2H), 7.58 (dd, J=18.1, 9.1 Hz, 2H), 7.07 (s, 1H), 6.41 (s, 2H), 5.68 (s, 1H), 4.85 (dq, J=13.7, 6.8 Hz, 1H), 4.70-4.58 (m, 1H), 4.35 (q, J=8.1 Hz, 1H), 4.17-4.07 (m, 1H), 3.97 (dd, J=14.2, 7.5 Hz, 2H), 3.74 (s, 1H), 3.69-3.57 (m, 1H), 3.56-3.42 (m, 2H), 3.42-3.13 (m, 4H), 2.96 (s, 2H), 2.93 (d, J=2.3 Hz, 1H), 2.89-2.76 (m, 4H), 2.66-2.53 (m, 7H), 2.42-2.15 (m, 2H), 2.10 (s, 3H), 2.07-1.98 (m, 1H), 1.87-1.55 (m, 8H), 1.46 (s, 6H), 1.44 (s, 9H), 1.34 (s, 9H), 0.87 (s, 9H), 0.09 (s, 3H), 0.05 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 187.07, 176.14, 174.46, 171.46, 171.04, 169.11, 166.93, 158.77, 156.39, 138.63, 133.66, 132.55, 124.65, 117.55, 105.24, 86.49, 82.64, 63.92, 63.84, 60.81, 57.07, 51.69, 51.31, 50.37, 49.56, 45.47, 43.51, 40.43, 37.55, 35.99, 30.28, 29.17, 28.83, 28.22, 26.11, 24.61, 19.49, 18.71, 18.19, 17.31, 12.69, −5.11, −5.44. MS (ESI) [MH]+ calcd. 1104.6. found 1104.6.
1H NMR (400 MHz, CDCl3) δ: 7.83 (d, J=8.1 Hz, 1H), 7.77 (s, 1H), 7.50 (d, J=21.3 Hz, 1H), 7.35 (m, 2H), 7.08 (dd, J=15.7, 7.5 Hz, 1H), 6.90 (s, 1H), 6.82 (s, 1H), 4.98 (d, J=27.6 Hz, 1H), 4.84-4.67 (m, 1H), 4.60 (s, 1H), 4.40-4.30 (m, 1H), 4.16-3.93 (m, 4H), 3.82 (s, 2H), 3.77-3.69 (m, 3H), 3.67 (d, J=6.3 Hz, 1H), 3.45-3.28 (m, 2H), 3.27-2.99 (m, 6H), 3.00-2.86 (m, 2H), 2.84 (s, 1H), 2.78-2.55 (m, 3H), 2.45-2.19 (m, 5H), 2.12 (s, 4H), 2.06-1.97 (m, 3H), 1.89 (d, J=4.7 Hz, 7H), 1.71-1.55 (m, 3H), 1.54-1.39 (m, 21H), 1.36 (s, 13H), 1.31-1.26 (m, 12H), 1.06 (t, J=10.5 Hz, 4H), 0.93-0.82 (m, 14H), 0.06 (s, 3H), 0.05 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 182.99, 175.20, 171.94, 170.75, 170.37, 168.93, 156.44, 77.43, 75.69, 66.32, 64.35, 63.93, 61.77, 57.52, 56.58, 54.28, 42.68, 40.37, 31.21, 30.95, 30.08, 29.88, 29.36, 28.85, 28.66, 26.01, 24.92, 24.16, 18.91, 18.48, 17.89, 15.48, −5.25, −5.29. MS (ESI) [MH]+ calcd. 983.6. found 983.6.
1H NMR (400 MHz, CDCl3) δ: 9.92 (s, 1H), 7.49 (d, J=4.8 Hz, 2H), 7.39-7.27 (m, 11H), 7.17-7.02 (m, 11H), 6.78 (d, J=8.4 Hz, 2H), 6.69 (s, 1H), 4.48 (p, J=6.9 Hz, 1H), 4.42-4.18 (m, 2H), 4.06-3.99 (m, 1H), 3.91 (dd, J=11.4, 3.8 Hz, 1H), 3.78 (dd, J=11.3, 4.4 Hz, 1H), 3.61 (dd, J=9.8, 4.8 Hz, 1H), 3.48-3.37 (m, 1H), 3.32-2.99 (m, 8H), 2.83 (dd, J=6.8, 3.4 Hz, 1H), 2.41-1.95 (m, 2H), 1.91-1.72 (m, 2H), 1.61 (s, 12H), 1.41 (s, 10H), 1.35-1.24 (m, 20H), 0.72 (s, 1H), 0.07 (s, 3H), 0.05 (s, 311). 13C NMR (101 MHz, CDCl3) δ: 184.31, 175.20, 172.61, 170.67, 169.47, 168.12, 154.53, 142.31, 138.19, 136.81, 131.91, 129.97, 129.85, 128.43, 128.37, 120.09, 77.43, 63.49, 62.17, 61.55, 57.84, 54.97, 52.19, 51.15, 48.67, 43.36, 42.53, 41.75, 36.36, 31.84, 30.99, 29.92, 29.88, 29.44, 29.05, 28.67, 26.07, 24.49, 19.80, 18.56, −5.19. MS (ESI) [MH]+ calcd. 866.5. found 866.5.
1H NMR (400 MHz, CDCl3) δ: 7.94 (t, J=9.8 Hz, 2H), 7.73 (d, J=8.7 Hz, 1H), 7.51 (t, J=6.7 Hz, 1H), 7.34-7.11 (m, 23H), 6.79 (s, 1H), 4.92-4.72 (m, 2H), 4.59 (dd, J=16.3, 8.6 Hz, 1H), 4.25 (d, J=11.5 Hz, 1H), 4.01 (d, J=10.7 Hz, 1H), 3.82 (dd, J=14.2, 7.6 Hz, 1H), 3.73 (s, 1H), 3.59-3.51 (m, 2H), 3.48-3.24 (m, 3H), 3.09 (dd, J=13.8, 8.9 Hz, 1H), 2.96-2.68 (m, 6H), 2.59 (dd, J=15.8, 3.6 Hz, 1H), 2.45-2.08 (m, 2H), 2.07-1.96 (m, 1H), 1.62 (s, 5H), 1.52-1.17 (m, 18H), 0.94 (s, 9H), 0.15 (s, 3H), 0.13 (s, 3H). 13C NMR (101 MHz, CDCl3) δ: 184.06, 175.64, 173.93, 171.01, 170.49, 169.15, 167.20, 144.28, 136.98, 129.61, 128.81, 128.49, 128.30, 127.48, 126.75, 77.55, 77.23, 76.91, 71.17, 63.63, 60.72, 57.12, 52.96, 51.54, 50.58, 49.71, 49.27, 45.35, 44.53, 37.27, 36.80, 33.93, 30.22, 29.24, 26.24, 24.32, 18.78, 17.89, 0.22, −5.11, −5.34. MS (ESI) [MH]+ calcd. 1027.5. found 1027.6.
1H NMR (CDCl3, 400 MHz) δ: 8.33 (bs, 1H), 8.17 (d, J=9.1 Hz, 1H), 8.03 (d, J=7.4 Hz, 1H), 7.54 (s, 1H), 7.21 (m, 5H), 6.04 (bs, 1H), 5.08 (bs, 1H), 4.58 (m, 1H), 3.87 (dd, J=8.1 Hz, 5.0 Hz, 1H), 3.80 (bs, 1H), 2.48 (dd, J=3.6 Hz, 1.8 Hz, 2H), 2.16 (m, 2H), 1.62-1.57 (m, 7H), 1.48 (m, 2H), 1.42 (m, 2H), 1.35 (m, 2H), 1.20 (m, 9H), 1.07 (m, 2H), 0.81 (m, 6H) ppm. MS (ESI) [MH]+ calcd. 626.8. found 626.3 and 313.7 for [M+2H]2+/2.
Cyclic peptide 22 was reacted as a crude material to yield conjugated peptide 23. (see page 39)1H NMR (300 MHz, DMSO) δ 8.95 (s, 1H), 8.68 (d, J=8.2 Hz, 1H), 8.32 (t, J=6.3 Hz, 1H), 7.68 (d, J=8.4 Hz, 1H), 7.51 (d, J=9.4 Hz, 1H), 7.42-7.08 (m, 5H), 6.46 (s, 1H), 6.31 (s, 1H), 4.47-4.15 (m, 3H), 3.73 (dd, J=20.5, 14.1 Hz, 3H), 3.45-3.33 (m, 2H), 3.17 (dd, J=20.9, 13.0 Hz, 2H), 2.98-2.64 (m, 4H), 2.40 (s, 2H), 2.22-2.10 (m, 1H), 1.91 (ddd, J=22.9, 13.4, 6.6 Hz, 2H), 1.79-1.40 (m, 5H), 1.30-1.15 (m, 9H), 0.93-0.74 (m, 6H). MS (ESI) [MH]+ calcd. 818.4. found 818.3.
HATU-Mediated Cyclization
Advantageously, the cyclization reaction can be run at least at 0.002M concentration of the starting amino acid/peptide. Preferably the concentration is run between 0.002M to 0.2M.
In another embodiment the process is run at a concentration of at least 0.1M of the starting amino acid/peptide. In another embodiment the process is run at a concentration of around 0.2M of the starting amino acid/peptide.
Traditionally, prior macrocyclizations cannot be performed at such high concentrations. This was demonstrated using a direct comparison with HATU process which fails in that the major products observed are derived from oligomerization and polymerization.
O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (“HATU”)-mediated cyclization of Pro-Gly-Gly-Gly was compared with the amino aldehyde/isocyanide cyclization method at 0.2 M. Upon completion, crude reaction mixtures were analyzed using liquid chromatography electrospray ionization mass spectrometry (“LC-MS(ESI)”) to assess the level of selectivity for the desired cyclic peptide.
Mass spectrometry (“MS”)-analysis of the HATU reaction revealed that cyclodimerization predominated at 0.2M (
Results from the MS peak analysis found 6 components.
Component 1: Peak at Scan 27.4. Top ions are 655 566 923
Component 2: Peak at Scan 28.4. Top ions are 982 744 714
Component 3: Peak at Scan 29.9. Top ions are 235 848 538
Component 4: Peak at Scan 30.9. Top ions are 559 827 560
Component 5: Peak at Scan 31.9. Top ions are 752 931
Component 6: Peak at Scan 32.7. Top ions are 537 295 446
The crude LC-MS(ESI) analysis for the isocyanide/amino aldehyde mediated cyclization revealed a high level of selectivity for the desired cyclic peptide (m/z=479) at 0.2M (
Results from the MS peak analysis found 2 components.
Component 1: Peak at Scan 29.7. Top ions are 287 212 383 (Pro-Gly-Gly-Gly)
Component 2: Peak at Scan 30.9. Top ions are 479 396 240 (cyclic peptide)
Method A: ring-opening of aziridine-containing cyclic peptide is conducted using an aromatic thiol as the nucleophile (Method A). In a screw-cap vial equipped with a magnetic stirring bar was added aziridine-containing cyclic peptide (0.06 mmol) and aromatic thiol (0.066 mmol) were added to 0.2 ml of degassed CHCl3. NEt3 (0.06 mmol) was then added to the solution and the reaction was stirred at room temperature for 3-4 hours. The reaction was then diluted with CH2Cl2, and washed twice with saturated aqueous NH4Cl (3 ml) followed by one wash with brine (3 ml). The organic layer was subsequently dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude ring-opened products were of high purity, but analytically pure samples were obtained following purification by flash column chromatography (5% MeOH in EtOAc).
Method B: ring-opening of aziridine-containing cyclic peptide is carried out using an aliphatic thiol or imide as the nucleophile (Method B). In a screw-cap vial equipped with a magnetic stirring bar was added aziridine-containing cyclic peptide (0.06 mmol) and aliphatic thiol or imide (0.066 mmol) to 0.2 ml of degassed CHCl3. DBU (0.06 mmol) was then added to the solution and the reaction was stirred at room temperature for 3-4 hours. The reaction was then diluted with CH2Cl2, and washed twice with saturated aqueous NH4Cl (3 ml) followed by one wash with brine (3 ml). The organic layer was subsequently dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude ring-opened products were of high purity, but analytically pure samples were obtained following purification by flash column chromatography (5% MeOH in EtOAc).
Results
Side chains including, but not limited to, fluorescent substituents have been appended to the products of macrocyclization (Scheme 3).
##STR00026##
The incorporation of an activated aziridine ring into the framework of a cyclic peptide provides a useful point for conjugation to various side-chains via nucleophilic ring-opening, a well developed methodology that has been demonstrated using nucleophilic biomolecules ranging from carbohydrates to biotin and farnesyl derivatives.14 As an example, cyclic peptide conjugation strategy can be demonstrated through the nucleophilic ring-opening of an aziridine moiety with the widely used fluorescent tag 7-mercapto-4-methyl-coumarin (7-mmc) (Scheme 3). In another aspect, a late-stage synthesis of a thioester residue by ring-opening of the cyclic peptide with commercially available thiobenzoic acid can also be used. The reaction proceeded smoothly, delivering the ring opened product in 98% yield. The late-stage incorporation of the thioester functionality avoids steps typically required for its protection from both oxidation and electrophilic reagents. Many other amino acid side chains, both natural and unnatural, can be installed by similar ring-opening protocols, opening doors for conformational optimization of cyclic peptides at a late stage of synthesis. Other nucleophiles such as aliphatic thiols, acids, secondary amines such as morpholine, and imides worked equally well during ring-opening.
In another aspect, cyclic peptides equipped with aziridine rings are subjected to ring-opening (
These data suggest that strong ion pairing (ΔpKa>1) between the base and nucleophile may result in decreased reactivity. The optimized conditions for ring-opening were found to proceed cleanly to afford the modified cyclic peptides in high purity following extractive work-up (
X-ray analysis of cyclic product 1 was conducted.
Results
##STR00027##
TABLE 4
Crystal data and structure refinement for k07227.
Identification code
k07227
Empirical formula
C24H38N3O3Si
Formula weight
445.67
Temperature
150(2) K
Wavelength
0.71073 Å
Crystal system
Monoclinic
Space group
P 21
Unit cell dimensions
a = 14.590(2) Å
α = 90°.
b = 12.326(2) Å
β = 94.718(7)°.
c = 15.088(2) Å
γ = 90°.
Volume
2704.2(7) Å3
Z
4
Density (calculated)
1.097 Mg/m3
Absorption coefficient
0.115 mm−1
F(000)
968
Crystal size
0.32 × 0.10 × 0.08 mm3
Theta range for data collection
2.62 to 23.05°.
Index ranges
−15 <= h <= 15, −13 <= k <= 12,
−15 <= l <= 16
Reflections collected
10115
Independent reflections
3828 [R(int) = 0.1359]
Completeness to theta = 23.05°
95.9%
Absorption correction
Semi-empirical from equivalents
Max. and min. transmission
1.000 and 0.770
Refinement method
Full-matrix least-squares on F2
Data/restraints/parameters
3828/1/559
Goodness-of-fit on F2
1.044
Final R indices [I > 2 sigma(I)]
R1 = 0.0982, wR2 = 0.2560
R indices (all data)
R1 = 0.1715, wR2 = 0.3055
Largest diff. peak and hole
0.492 and −0.316 e · Å−3
TABLE 5
Atomic coordinates (×104) and equivalent isotropic displacement
parameters (Å2 × 103) for k07227. U(eq) is defined as
one third of the trace of the orthogonalized Uij tensor.
x
y
z
U(eq)
Si(1A)
7245(3)
4768(4)
8396(3)
68(1)
O(1A)
3310(7)
4696(12)
6317(8)
94(4)
O(2A)
4778(6)
4429(8)
4729(7)
62(3)
O(3A)
4070(7)
3807(11)
8551(7)
82(3)
O(4A)
6881(6)
4731(9)
7332(6)
64(3)
N(1A)
5222(7)
4298(9)
6198(7)
53(3)
N(2A)
2735(8)
3545(12)
7673(8)
74(4)
C(1A)
4577(10)
4187(12)
5473(9)
56(4)
C(2A)
3624(9)
3854(14)
5700(9)
61(4)
C(3A)
3884(8)
4758(14)
7161(9)
61(4)
C(4A)
4916(9)
4610(11)
7073(9)
57(4)
C(5A)
5474(9)
5392(12)
6570(9)
55(4)
C(6A)
6464(10)
5591(15)
6823(10)
72(5)
C(7A)
7044(16)
3423(18)
8820(12)
118(8)
C(8A)
6568(12)
5820(20)
9009(13)
105(6)
C(9A)
8501(12)
5097(14)
8489(14)
86(6)
C(10A)
8607(13)
6160(30)
8022(19)
150(11)
C(11A)
9045(13)
4350(30)
8043(17)
169(14)
C(12A)
8846(14)
5210(20)
9454(15)
133(9)
C(13A)
3590(10)
3973(17)
7871(11)
76(5)
C(14A)
2268(10)
2780(16)
8263(10)
74(5)
C(15A)
2819(13)
1767(18)
8419(13)
103(6)
C(16A)
2093(11)
3415(16)
9107(10)
86(5)
C(17A)
1325(12)
2540(20)
7748(12)
110(7)
C(18A)
2957(9)
3707(15)
4897(9)
66(4)
C(19A)
2151(10)
3006(18)
5091(10)
77(5)
C(20A)
2294(13)
1877(19)
5232(12)
90(5)
C(21A)
1548(17)
1230(20)
5361(14)
112(7)
C(22A)
725(19)
1640(30)
5502(16)
128(9)
C(23A)
525(13)
2770(30)
5385(13)
116(9)
C(24A)
1270(12)
3480(20)
5217(11)
101(7)
Si(1B)
8204(3)
2344(4)
2871(3)
68(1)
O(1B)
6478(7)
2195(11)
6755(7)
86(3)
O(2B)
4724(6)
1912(7)
5174(6)
53(2)
O(3B)
8659(6)
1106(10)
6205(7)
75(3)
O(4B)
7241(6)
2259(8)
3360(6)
66(3)
N(1B)
6249(7)
1815(8)
4890(7)
48(3)
N(2B)
7884(8)
963(11)
7449(9)
69(3)
C(1B)
5494(10)
1740(12)
5431(9)
55(4)
C(2B)
5816(9)
1389(12)
6398(9)
53(3)
C(3B)
7344(9)
2232(14)
6288(8)
59(4)
C(4B)
7165(9)
2098(12)
5256(9)
60(4)
C(5B)
6589(10)
2897(14)
4693(11)
70(4)
C(6B)
6790(11)
3147(14)
3790(11)
74(5)
C(7B)
8766(12)
968(16)
3042(13)
97(6)
C(8B)
8926(11)
3466(19)
3390(11)
98(6)
C(9B)
7893(10)
2620(14)
1666(10)
71(5)
C(10B)
7510(13)
3777(14)
1548(11)
85(5)
C(11B)
7165(14)
1807(19)
1279(12)
103(6)
C(12B)
8748(12)
2536(19)
1143(11)
100(6)
C(13B)
8015(10)
1332(15)
6630(11)
69(4)
C(14B)
8453(10)
163(13)
7952(11)
66(4)
C(15B)
8439(13)
−842(17)
7445(15)
111(7)
C(16B)
9438(11)
564(17)
8135(13)
97(6)
C(17B)
8040(11)
0(20)
8825(11)
103(7)
C(18B)
5021(9)
1225(13)
6976(9)
61(4)
C(19B)
5242(8)
526(15)
7787(10)
65(5)
C(20B)
5337(10)
−610(14)
7633(11)
65(4)
C(21B)
5477(11)
−1260(20)
8343(13)
101(7)
C(22B)
5524(12)
−940(20)
9202(18)
105(7)
C(23B)
5439(11)
130(30)
9374(13)
100(7)
C(24B)
5292(10)
878(19)
8643(11)
86(6)
TABLE 6
Bond lengths [Å] and angles [°] for k07227.
Si(1A)—O(4A)
1.649(10)
Si(1A)—C(7A)
1.81(2)
Si(1A)—C(9A)
1.871(18)
Si(1A)—C(8A)
1.91(2)
O(1A)—C(3A)
1.468(17)
O(1A)—C(2A)
1.491(18)
O(2A)—C(1A)
1.221(15)
O(3A)—C(13A)
1.211(17)
O(4A)—C(6A)
1.418(18)
N(1A)—C(1A)
1.390(17)
N(1A)—C(4A)
1.479(16)
N(1A)—C(5A)
1.494(18)
N(2A)—C(13A)
1.36(2)
N(2A)—C(14A)
1.50(2)
C(1A)—C(2A)
1.52(2)
C(2A)—C(18A)
1.502(19)
C(3A)—C(13A)
1.53(2)
C(3A)—C(4A)
1.533(18)
C(4A)—C(5A)
1.507(19)
C(5A)—C(6A)
1.48(2)
C(9A)—C(11A)
1.42(3)
C(9A)—C(10A)
1.50(3)
C(9A)—C(12A)
1.51(3)
C(14A)—C(15A)
1.49(3)
C(14A)—C(16A)
1.53(2)
C(14A)—C(17A)
1.55(2)
C(18A)—C(19A)
1.51(2)
C(19A)—C(20A)
1.42(3)
C(19A)—C(24A)
1.44(2)
C(20A)—C(21A)
1.38(3)
C(21A)—C(22A)
1.34(3)
C(22A)—C(23A)
1.44(4)
C(23A)—C(24A)
1.44(3)
Si(1B)—O(4B)
1.643(9)
Si(1B)—C(9B)
1.868(16)
Si(1B)—C(8B)
1.871(19)
Si(1B)—C(7B)
1.893(18)
O(1B)—C(2B)
1.457(18)
O(1B)—C(3B)
1.496(16)
O(2B)—C(1B)
1.177(15)
O(3B)—C(13B)
1.214(16)
O(4B)—C(6B)
1.457(18)
N(1B)—C(1B)
1.428(17)
N(1B)—C(4B)
1.446(17)
N(1B)—C(5B)
1.461(19)
N(2B)—C(13B)
1.344(19)
N(2B)—C(14B)
1.461(19)
C(1B)—C(2B)
1.56(2)
C(2B)—C(18B)
1.522(18)
C(3B)—C(13B)
1.54(2)
C(3B)—C(4B)
1.567(18)
C(4B)—C(5B)
1.51(2)
C(5B)—C(6B)
1.45(2)
C(9B)—C(12B)
1.53(2)
C(9B)—C(10B)
1.54(2)
C(9B)—C(11B)
1.54(2)
C(14B)—C(15B)
1.45(3)
C(14B)—C(17B)
1.51(2)
C(14B)—C(16B)
1.52(2)
C(18B)—C(19B)
1.51(2)
C(19B)—C(24B)
1.36(2)
C(19B)—C(20B)
1.43(2)
C(20B)—C(21B)
1.34(2)
C(21B)—C(22B)
1.35(3)
C(22B)—C(23B)
1.35(3)
C(23B)—C(24B)
1.44(3)
O(4A)—Si(1A)—C(7A)
105.7(8)
O(4A)—Si(1A)—C(9A)
108.3(7)
C(7A)—Si(1A)—C(9A)
111.0(9)
O(4A)—Si(1A)—C(8A)
110.6(8)
C(7A)—Si(1A)—C(8A)
110.1(10)
C(9A)—Si(1A)—C(8A)
111.0(9)
C(3A)—O(1A)—C(2A)
113.3(11)
C(6A)—O(4A)—Si(1A)
126.6(10)
C(1A)—N(1A)—C(4A)
119.6(10)
C(1A)—N(1A)—C(5A)
121.0(11)
C(4A)—N(1A)—C(5A)
60.9(8)
C(13A)—N(2A)—C(14A)
124.4(13)
O(2A)—C(1A)—N(1A)
120.1(13)
O(2A)—C(1A)—C(2A)
124.6(12)
N(1A)—C(1A)—C(2A)
115.0(11)
O(1A)—C(2A)—C(18A)
112.0(12)
O(1A)—C(2A)—C(1A)
106.7(12)
C(18A)—C(2A)—C(1A)
113.3(11)
O(1A)—C(3A)—C(13A)
113.7(13)
O(1A)—C(3A)—C(4A)
114.4(11)
C(13A)—C(3A)—C(4A)
108.6(12)
N(1A)—C(4A)—C(5A)
60.0(8)
N(1A)—C(4A)—C(3A)
118.7(11)
C(5A)—C(4A)—C(3A)
122.9(12)
C(6A)—C(5A)—N(1A)
116.8(12)
C(6A)—C(5A)—C(4A)
122.1(12)
N(1A)—C(5A)—C(4A)
59.1(8)
O(4A)—C(6A)—C(5A)
112.4(12)
C(11A)—C(9A)—C(10A)
105(2)
C(11A)—C(9A)—C(12A)
111.3(18)
C(10A)—C(9A)—C(12A)
109(2)
C(11A)—C(9A)—Si(1A)
113.9(16)
C(10A)—C(9A)—Si(1A)
107.0(12)
C(12A)—C(9A)—Si(1A)
109.9(13)
O(3A)—C(13A)—N(2A)
125.1(16)
O(3A)—C(13A)—C(3A)
121.6(14)
N(2A)—C(13A)—C(3A)
113.2(14)
C(15A)—C(14A)—N(2A)
110.8(13)
C(15A)—C(14A)—C(16A)
114.8(16)
N(2A)—C(14A)—C(16A)
106.7(15)
C(15A)—C(14A)—C(17A)
111.4(17)
N(2A)—C(14A)—C(17A)
104.4(13)
C(16A)—C(14A)—C(17A)
108.1(14)
C(2A)—C(18A)—C(19A)
112.2(12)
C(20A)—C(19A)—C(24A)
120.2(19)
C(20A)—C(19A)—C(18A)
118.9(16)
C(24A)—C(19A)—C(18A)
121(2)
C(21A)—C(20A)—C(19A)
119(2)
C(22A)—C(21A)—C(20A)
122(3)
C(21A)—C(22A)—C(23A)
122(3)
C(22A)—C(23A)—C(24A)
118(2)
C(23A)—C(24A)—C(19A)
118(2)
O(4B)—Si(1B)—C(9B)
107.5(6)
O(4B)—Si(1B)—C(8B)
109.4(7)
C(9B)—Si(1B)—C(8B)
110.9(8)
O(4B)—Si(1B)—C(7B)
105.0(7)
C(9B)—Si(1B)—C(7B)
111.3(9)
C(8B)—Si(1B)—C(7B)
112.5(9)
C(2B)—O(1B)—C(3B)
114.0(11)
C(6B)—O(4B)—Si(1B)
125.9(10)
C(1B)—N(1B)—C(4B)
122.0(11)
C(1B)—N(1B)—C(5B)
117.7(11)
C(4B)—N(1B)—C(5B)
62.6(9)
C(13B)—N(2B)—C(14B)
126.0(13)
O(2B)—C(1B)—N(1B)
124.1(13)
O(2B)—C(1B)—C(2B)
124.4(12)
N(1B)—C(1B)—C(2B)
111.4(11)
O(1B)—C(2B)—C(18B)
113.1(11)
O(1B)—C(2B)—C(1B)
107.3(11)
C(18B)—C(2B)—C(1B)
112.9(11)
O(1B)—C(3B)—C(13B)
111.0(12)
O(1B)—C(3B)—C(4B)
112.7(10)
C(13B)—C(3B)—C(4B)
107.8(12)
N(1B)—C(4B)—C(5B)
59.2(8)
N(1B)—C(4B)—C(3B)
118.6(10)
C(5B)—C(4B)—C(3B)
122.0(13)
C(6B)—C(5B)—N(1B)
118.9(14)
C(6B)—C(5B)—C(4B)
121.4(13)
N(1B)—C(5B)—C(4B)
58.2(9)
C(5B)—C(6B)—O(4B)
113.1(13)
C(12B)—C(9B)—C(10B)
107.8(14)
C(12B)—C(9B)—C(11B)
109.0(15)
C(10B)—C(9B)—C(11B)
109.1(14)
C(12B)—C(9B)—Si(1B)
110.2(11)
C(10B)—C(9B)—Si(1B)
109.8(12)
C(11B)—C(9B)—Si(1B)
110.8(12)
O(3B)—C(13B)—N(2B)
125.6(15)
O(3B)—C(13B)—C(3B)
119.1(14)
N(2B)—C(13B)—C(3B)
114.7(13)
C(15B)—C(14B)—N(2B)
108.6(13)
C(15B)—C(14B)—C(17B)
110.9(16)
N(2B)—C(14B)—C(17B)
107.0(13)
C(15B)—C(14B)—C(16B)
110.2(15)
N(2B)—C(14B)—C(16B)
111.3(13)
C(17B)—C(14B)—C(16B)
108.9(15)
C(19B)—C(18B)—C(2B)
114.7(11)
C(24B)—C(19B)—C(20B)
118.0(16)
C(24B)—C(19B)—C(18B)
125.4(18)
C(20B)—C(19B)—C(18B)
116.5(14)
C(21B)—C(20B)—C(19B)
117.9(18)
C(20B)—C(21B)—C(22B)
125(3)
C(23B)—C(22B)—C(21B)
119(2)
C(22B)—C(23B)—C(24B)
119(2)
C(19B)—C(24B)—C(23B)
121(2)
TABLE 7
Anisotropic displacement parameters (Å2 × 103) for k07227.
The anisotropic displacement factor exponent takes the
form: −2π2[h2 a*2U11 + . . . + 2 h k a* b* U12]
U11
U22
U33
U23
U13
U12
Si(1A)
59(2)
75(3)
67(3)
−5(2)
−2(2)
−7(2)
O(1A)
67(6)
116(10)
101(8)
−29(8)
23(6)
−1(7)
O(2A)
66(6)
55(6)
64(7)
−6(5)
11(5)
0(5)
O(3A)
61(6)
118(10)
66(7)
14(7)
8(5)
−7(6)
O(4A)
55(5)
64(6)
76(6)
5(6)
11(4)
−3(5)
N(1A)
54(7)
54(7)
54(7)
−6(6)
19(6)
1(6)
N(2A)
51(7)
99(11)
74(8)
17(8)
12(6)
6(7)
C(1A)
77(10)
49(9)
40(8)
1(7)
−2(7)
3(7)
C(2A)
60(9)
70(10)
52(8)
−9(8)
3(7)
6(8)
C(3A)
44(7)
70(10)
70(9)
−7(9)
5(6)
7(7)
C(4A)
61(8)
46(9)
65(9)
9(7)
7(7)
0(7)
C(5A)
51(8)
48(9)
64(9)
4(7)
4(7)
4(7)
C(6A)
62(10)
86(13)
69(10)
20(10)
18(8)
−7(9)
C(7A)
170(20)
110(17)
71(12)
20(12)
−25(12)
−17(16)
C(8A)
83(12)
124(18)
109(14)
−12(13)
23(10)
3(12)
C(9A)
69(10)
59(11)
130(16)
44(11)
17(11)
11(9)
C(10A)
47(11)
180(30)
220(30)
30(20)
−10(13)
−16(14)
C(11A)
61(13)
300(50)
140(20)
−60(20)
−2(13)
13(19)
C(12A)
101(14)
170(20)
122(17)
57(17)
−40(13)
−18(15)
C(13A)
49(9)
122(15)
59(9)
−5(10)
12(8)
−2(10)
C(14A)
58(9)
101(14)
64(10)
15(9)
17(7)
−2(9)
C(15A)
95(14)
103(16)
113(15)
37(13)
27(11)
15(12)
C(16A)
85(11)
105(15)
68(11)
−27(10)
17(8)
−17(10)
C(17A)
82(12)
150(20)
95(13)
10(14)
15(10)
−60(13)
C(18A)
63(9)
86(12)
51(8)
3(8)
8(7)
−16(9)
C(19A)
59(10)
122(18)
49(9)
5(10)
−2(7)
2(10)
C(20A)
97(13)
92(15)
85(12)
4(11)
31(10)
−19(12)
C(21A)
118(18)
121(19)
100(15)
8(13)
21(13)
−48(16)
C(22A)
95(19)
190(30)
102(17)
11(19)
0(13)
−26(19)
C(23A)
50(11)
210(30)
86(14)
9(17)
1(9)
1(16)
C(24A)
63(12)
170(20)
68(11)
−7(13)
−7(8)
0(13)
Si(1B)
60(3)
75(3)
70(3)
8(3)
13(2)
2(2)
O(1B)
71(7)
106(9)
83(7)
14(7)
14(5)
3(7)
O(2B)
51(6)
44(5)
65(6)
−3(5)
3(4)
3(4)
O(3B)
52(6)
99(9)
75(7)
4(6)
9(5)
9(6)
O(4B)
69(6)
60(6)
71(6)
11(6)
24(5)
5(5)
N(1B)
60(7)
36(6)
49(6)
−1(5)
6(5)
13(5)
N(2B)
50(7)
75(9)
84(9)
10(8)
13(6)
3(7)
C(1B)
57(10)
48(9)
61(9)
−17(7)
6(7)
−4(7)
C(2B)
45(8)
50(9)
65(9)
−11(7)
4(6)
0(7)
C(3B)
51(8)
72(10)
54(8)
−1(8)
3(6)
5(8)
C(4B)
47(8)
62(10)
73(9)
25(8)
17(7)
8(7)
C(5B)
59(9)
65(11)
86(12)
6(9)
8(8)
−9(8)
C(6B)
82(11)
66(11)
77(11)
15(9)
21(8)
17(9)
C(7B)
84(12)
87(14)
123(15)
34(12)
28(10)
36(11)
C(8B)
64(10)
147(19)
84(12)
−19(13)
10(8)
−22(11)
C(9B)
56(9)
78(13)
82(11)
10(9)
14(8)
−12(8)
C(10B)
113(13)
64(11)
77(11)
23(10)
11(9)
−7(10)
C(11B)
121(16)
104(16)
84(12)
−14(12)
11(11)
−22(13)
C(12B)
97(13)
117(17)
89(12)
32(13)
36(10)
13(12)
C(13B)
48(9)
85(12)
75(11)
−3(10)
18(8)
−9(8)
C(14B)
66(10)
50(10)
81(11)
11(9)
3(8)
0(8)
C(15B)
88(13)
88(17)
152(19)
37(15)
−11(12)
29(11)
C(16B)
62(10)
112(17)
116(15)
14(13)
−1(10)
−1(10)
C(17B)
80(11)
150(20)
74(11)
55(13)
12(9)
23(12)
C(18B)
48(8)
73(11)
61(9)
−5(8)
7(6)
6(7)
C(19B)
34(7)
105(15)
56(10)
14(10)
8(6)
−16(8)
C(20B)
67(10)
64(11)
64(10)
11(9)
3(7)
1(8)
C(21B)
76(12)
150(20)
74(13)
14(16)
6(10)
−11(12)
C(22B)
67(12)
120(20)
120(20)
35(17)
13(11)
−20(13)
C(23B)
60(11)
180(30)
64(12)
9(16)
10(8)
−26(14)
C(24B)
59(10)
138(17)
63(11)
−3(12)
14(8)
−8(10)
TABLE 8
Hydrogen coordinates (×104) and isotropic displacement
parameters (Å2 × 103) for k07227.
x
y
z
U (eq)
H(2A)
2439
3731
7164
89
H(2AB)
3677
3148
6028
73
H(3AA)
3805
5507
7399
73
H(4AA)
5261
4254
7594
69
H(5AA)
5126
6016
6284
66
H(6AA)
6788
5688
6277
86
H(6AB)
6530
6272
7171
86
H(7AA)
7391
2890
8499
177
H(7AB)
6386
3254
8734
177
H(7AC)
7246
3395
9455
177
H(8AA)
5915
5621
8958
157
H(8AB)
6644
6535
8745
157
H(8AC)
6797
5834
9638
157
H(10A)
9261
6348
8041
225
H(10B)
8274
6725
8319
225
H(10C)
8356
6093
7402
225
H(11A)
9691
4578
8109
254
H(11B)
8832
4327
7410
254
H(11C)
8989
3625
8301
254
H(12A)
9506
5375
9499
199
H(12B)
8742
4534
9768
199
H(12C)
8516
5804
9723
199
H(15A)
3407
1942
8750
154
H(15B)
2933
1440
7847
154
H(15C)
2478
1254
8764
154
H(16A)
2680
3561
9449
128
H(16B)
1699
2986
9469
128
H(16C)
1788
4103
8942
128
H(17A)
1421
2134
7206
166
H(17B)
1014
3229
7588
166
H(17C)
944
2115
8123
166
H(18A)
3279
3371
4415
80
H(18B)
2729
4426
4688
80
H(20A)
2894
1576
5238
108
H(21A)
1622
461
5349
135
H(22A)
261
1165
5684
154
H(23A)
−81
3040
5417
140
H(24A)
1185
4248
5190
122
H(2B)
7412
1227
7705
83
H(2BB)
6146
681
6365
64
H(3BA)
7647
2950
6413
71
H(4BA)
7677
1752
4955
72
H(5BA)
6321
3505
5026
84
H(6BA)
7190
3796
3799
89
H(6BB)
6209
3326
3435
89
H(7BA)
8930
851
3678
146
H(7BB)
9322
938
2721
146
H(7BC)
8336
402
2817
146
H(8BA)
8613
4161
3276
147
H(8BB)
9522
3478
3134
147
H(8BC)
9023
3346
4033
147
H(10D)
7339
3913
916
127
H(10E)
7981
4300
1769
127
H(10F)
6967
3855
1883
127
H(11D)
7007
1971
649
154
H(11E)
6613
1863
1604
154
H(11F)
7412
1069
1336
154
H(12D)
8576
2679
512
149
H(12E)
9009
1805
1211
149
H(12F)
9206
3070
1372
149
H(15D)
8712
−718
6882
166
H(15E)
7802
−1087
7323
166
H(15F)
8792
−1398
7788
166
H(16D)
9719
649
7571
146
H(16E)
9792
36
8509
146
H(16F)
9437
1264
8442
146
H(17D)
7413
−280
8715
154
H(17E)
8026
694
9140
154
H(17F)
8414
−521
9189
154
H(18C)
4814
1945
7173
73
H(18D)
4502
892
6609
73
H(20B)
5304
−894
7046
78
H(21B)
5548
−2016
8234
121
H(22B)
5615
−1446
9672
126
H(23B)
5475
386
9970
120
H(24B)
5229
1630
8761
104
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All references mentioned herein are incorporated by reference in their entirety.
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