A topographically and conformationally constrained,spin‐labeled, α‐amino acid: crystallographic characterization in peptides*

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C^α‐tetrasubstituted α‐amino acid. Here, we describe the molecular and crystal structures, as determined by X‐ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)₂·1,1,1,3,3,3‐hexafluoropropan‐2‐ol (HFIP) solvate, and five TOAC‐containing, terminally protected, linear peptides ranging in length from tetra‐ to hepta‐peptides. Incipient and fully developed, regular or distorted 3₁₀‐helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X‐ray diffraction structure of an intramolecularly cyclized side product resulting from a C‐activated TOAC residue has also been determined.     

Synthesis of stable isotopes of auxinic herbicides 4‐amino‐3,5,6‐trichloropicolinic acid and 4‐amino‐3,6‐dichloropicolinic acid

Pentachloropyridine serves as a key intermediate in the synthesis of 4‐amino‐3,5,6‐trichloropicolinic acid (picloram) and 4‐amino‐3,6‐dichloropicolinic acid (aminopyralid). An M+3 stable isotope of pentachloropyridine (1, pentachloropyridine‐1‐¹⁵N‐2,6‐¹³C₂) was prepared from K¹³C¹⁵N. Isotopically labeled pentachloropyridine was then carried through a seven‐step synthesis to give an M+3 stable isotope of 4‐amino‐3,5,6‐trichloropicolinic acid (2, picloram‐1‐¹⁵N‐2,6‐¹³C₂) in an overall yield of 42%. The chlorine atom in the 5‐position of 2 was selectively removed via electrochemical reduction. Carrying out the electrochemical reduction in water provided an M+3 stable isotope of 4‐amino‐3,6‐dichloropicolinic acid (3, aminopyralid‐1‐¹⁵N‐2,6‐¹³C₂), whereas conducting the reduction in deuterium oxide produced an M+4 stable isotope (4, aminopyralid‐1‐¹⁵N‐2,6‐¹³C₂‐5‐²H). Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of deuterium‐labeled 3‐O‐methyldopa and 4‐O‐methyldopa

Concise methods for the synthesis of 4‐hydroxy‐3‐[²H₃]‐methoxyphenylalanine (3‐O‐[²H₃]‐methydopa) and 3‐hydroxy‐4‐[²H₃]‐methoxyphenylalanine (4‐O‐[²H₃]‐methydopa) are described. The 3‐O‐[²H₃]‐methydopa is a valuable internal standard for the tandem MS quantification of 3‐O‐methyldopa, a metabolite of value in the diagnosis of aromatic l‐amino acid decarboxylase (AADC) deficiency. Copyright © 2003 John Wiley & Sons, Ltd.     

Highly efficient preparation of carbon‐14 labeled,auxin herbicide 4‐amino‐3,5,6‐trichloropicolinic acid

A highly efficient, seven‐step route has been developed for the preparation of 2,6‐[¹⁴C₂]‐4‐amino‐3,5,6‐trichloropicolinic acid ([¹⁴C₂]‐ 1 , 2,6‐[¹⁴C₂]‐picloram) from 2,6‐[¹⁴C₂]‐pentachloropyridine. The method involves the stepwise, highly selective and high yield introduction of amino and carboxylic acid groups to the 4‐ and 2‐positions, respectively, of pentachloropyridine affording [¹⁴C₂]‐ 1 in an overall radiochemical yield of >70% with the use of only one formal purification step. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of dermorphin‐(1–4) derivatives catalyzed by proteases in organic solvents*

Abstract: In order to extend the use of proteases to organic synthesis and seek the rules of enzymatic reactions in organic media, we focused on unnatural substrates for proteases to form amide bonds. In this paper, the study of unnatural substrates containing d ‐amino acid residue, which act as acyl acceptors as well as acyl donors for proteases in organic media, is reported. Dermorphin is a heptapeptide (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂) with potent analgesic activity. The N‐terminal tetrapeptide is the minimum sequence that retains dermorphin activity, and is selected as the model compound in our study. Two dermorphin‐(1–4) derivatives, Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐N₂H₂Ph and Boc‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which contained a d ‐amino acid residue, were synthesized by proteases in organic media for the first time. The synthesis of these two dermorphin‐(1–4) derivatives could be catalyzed by subtilisin with Boc‐Tyr‐d ‐Ala‐OCH₂CF₃ as an acyl donor substrate in AcOEt. The synthesis of dermorphin‐(1–2) derivative Boc‐Tyr‐d ‐Ala‐N₂H₂Ph was catalyzed by α‐chymotrypsin in different organic solvents and d ‐Ala‐N₂H₂Ph was used as an acyl acceptor substrate. Factors influencing the above enzymatic reactions were systematically studied.     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

Ac10c: a medium‐ring,cycloaliphatic Cα,α‐disubstituted glycine. Incorporation into model peptides and preferred conformation

Abstract: Two complete series of N‐protected oligopeptide esters to the pentamer level from 1‐amino‐cyclodecane‐1‐carboxylic acid (Ac₁₀c), an α‐amino acid conformationally constrained through a medium‐ring C^α_i ? C^α_i cyclization, and either the l ‐Ala or Aib residue, along with the N‐protected Ac₁₀c monomer and homo‐dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT‐IR absorption and ¹H NMR techniques. Furthermore, the molecular structures of two derivatives (Z‐Ac₁₀c‐OH and Fmoc‐Ac₁₀c‐OH) and two peptides (the dipeptide ester Z‐Ac₁₀c‐l ‐Phe‐OMe and the tripeptide ester Z‐Aib‐Ac₁₀c‐Aib‐OtBu) were determined in the crystal state using X‐ray diffraction. The experimental results support the view that β‐bends and 3₁₀‐helices are preferentially adopted by peptides rich in Ac₁₀c, the third largest cycloaliphatic C^α,α‐disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1‐amino‐cycloalkane‐1‐carboxylic acid (Ac_nc, with n = 3–12) series, which represents the prerequisite for our recent proposal of the ‘Ac_nc scan’ concept.     

Convenient and efficient synthesis of Boc‐/Z‐/Fmoc‐β‐amino acids employingN‐protected α‐amino acid fluorides

Abstract: A new and efficient method for the synthesis ofN^α‐Fmoc‐/Boc‐/Z‐β‐amino acids using the two‐step Arndt‐Eistert approach is described. Fmoc‐/Boc‐/Z‐α‐Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc‐/Boc‐/Z‐α‐aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding β‐amino acids using PhCOOAg/dioxane/H₂O.     

Synthesis of C‐14 and C‐13, H‐2‐labeled IKK inhibitor: [14C] and [13C4,D3]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido[3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide

[¹⁴C]‐N‐(6‐Chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5B ), an IKK inhibitor, was synthesized from [¹⁴C]‐barium carbonate in two steps in an overall radiochemical yield of 41%. The intermediate, [carboxyl‐¹⁴C]‐2‐methylnicotinic acid, was prepared by the lithiation and carbonation of 3‐bromo‐2‐methylpyridine. [¹³C₄,D₃]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5C ) was synthesized from [1,2,3,4‐¹³C₄]‐ethyl acetoacetate and [D₄]‐methanol in six steps in an overall yield of 2%. [¹³C₄]‐2‐methylnicotic acid, was prepared by condensation of [¹³C₄]‐ethyl 3‐aminocrotonate and acrolein, followed by hydrolysis with lithium hydroxide. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of 4‐thia[5‐13C]lysine

This report describes the synthesis of 4‐thia[5‐¹³C]lysine, an isotopomer of 4‐thialysine that is an analog of lysine. It was synthesized from 2‐amino[1‐¹³C]ethanol hydrochloride (1) in two steps. In the first step, 1 was converted to 2‐bromo[2‐¹³C]ethylamine hydrobromide (2). The reaction of cysteine with 2 in basic condition followed by acidification afforded 4‐thia[5‐¹³C]lysine hydrochloride (3).     

A simple convenient synthesis of l‐[4‐13C]glutamine

l ‐[4‐¹³C]Glutamine was synthesized from sodium [2‐¹³C]acetate in 12 steps and 18% overall yield. A Wittig reaction of (R)‐benzyl 4‐formyl‐2,2‐dimethyloxazolidine‐3‐carboxylate and ethyl 2‐(triphenylphosphoranylidene)[2‐¹³C]acetate prepared from d ‐serine and sodium [2‐¹³C]acetate, respectively, gave (4S)‐4‐(2‐ethoxycarbonyl[2‐¹³C]vinyl)‐2,2‐dimethyloxazolidine‐3‐carboxylic acid α,β‐isopropylidene group, oxidation of the resulting hydroxyl group to a carboxyl group and transamidation of the ester moiety gave l ‐N‐Cbz‐[4‐¹³C]glutamine (Cbz = benzyloxycarbonyl). Finally, removal of the Cbz group gave l ‐[4‐¹³C]glutamine. l ‐[4‐¹³C]Glutamine can be prepared in fewer steps and higher yield by this method compared with previously reported methods.     

Carbon‐14‐and carbon‐13‐labeled phosphoric acid [2‐4‐(4‐cyanophenyl)‐thiazol‐2‐yl]‐(2,4‐difluorophenyl)‐1‐[1,2,4]triazol‐4‐yl‐methylpropoxymethyl] monoester dilysine salt,a prodrug of ravuconazole

4‐Bromobenzoic acid [carboxyl‐¹⁴C] and 4‐(2‐bromoacetyl) [Ar‐¹³C₆]benzonitrile were transformed into the title compounds containing [ring¹⁴C‐thiazol‐4‐yl] and [Ar‐¹³C₆‐benzonitrile]. ¹⁴C‐Ravuconazole was prepared in 37% yield and Purity >99%. ¹³C₆‐Ravuconazole was made in 56% overall yield and Purity of >98%. Each labeled compound was converted by additional reaction steps to the corresponding labeled prodrug. Copyright © 2011 John Wiley & Sons, Ltd     

Synthesis of (±)‐6,6‐[2H6]dimethyl‐11‐nor‐Δ9‐tetrahydrocannabivarin‐9‐carboxylic acid

Starting from divarinol ( 4 ) using previously published procedures, (±)‐6,6‐[²H₆]Dimethyl‐11‐nor‐Δ⁹THCV‐9‐carboxylic acid ( 3 ) was synthesized for use as an internal standard in GC/MS analysis of 11‐nor‐Δ⁹THCV‐9‐carboxylic acid ( 2 ). The detection of 2 distinguishes the use of marijuana from the ingestion of Marinol^®. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of enantiopure non‐natural α‐amino acids using tert‐butyl (2S)‐2‐[bis‐(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate as key‐intermediate:the first synthesis of(S)‐2‐amino‐oleic acid

Abstract: A general method for the synthesis of enantiopure non‐natural α‐amino acids is described. The key intermediate tert‐butyl (2S)‐2‐[bis(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate was obtained from l ‐glutamic acid after suitable protection and selective reduction of the γ‐methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of δ,ε‐unsaturated α‐amino acids. This methodology was applied to the synthesis of (S)‐2‐amino‐oleic acid.     

Amino acid–azetidine chimeras: synthesis of enantiopure 3‐substituted azetidine‐2‐carboxylic acids

Abstract: Azetidine‐2‐carboxylic acid (Aze) analogs possessing various heteroatomic side chains at the 3‐position have been synthesized by modification of 1‐9‐(9‐phenylfluorenyl) (PhF)‐3‐allyl‐Aze tert‐butyl ester (2S,3S)‐ 1 . 3‐Allyl‐Aze 1 was synthesized by regioselective allylation of α‐tert‐butyl β‐methyl N‐(PhF)aspartate 13 , followed by selective ω‐carboxylate reduction, tosylation, and intramolecular N‐alkylation. Removal of the PhF group and olefin reduction by hydrogenation followed by Fmoc protection produced nor‐leucine–Aze chimera 2 . Regioselective olefin hydroboration of (2S,3S)‐ 1 produced primary alcohol 23 , which was protected as a silyl ether, hydrogenated and N‐protected to give 1‐Fmoc‐3‐hydroxypropyl‐Aze 26 . Enantiopure (2S,3S)‐3‐(3‐azidopropyl)‐1‐Fmoc‐azetidine‐2‐carboxylic acid tert‐butyl ester 3 was prepared as a Lys–Aze chimera by activation of 3‐hydroxypropyl–Aze 26 as a methanesulfonate and displacement with sodium azide. Moreover, orthogonally protected azetidine dicarboxylic acid 4 was synthesized as an α‐aminoadipate–Aze chimera by oxidation of alcohol 26 . These orthogonally protected amino acid–Aze chimeras are designed to serve as tools for studying the influence of conformation on peptide activity.     

Comparing hypoxia‐targeting potential of 99mTc(CO)3‐labeled 2‐nitro and 4‐nitroimidazole

Non‐invasive determination of hypoxia is an important problem in clinical nuclear medicine. Although ¹⁸F‐fluoromisonidazole is used clinically for hypoxia determination, the short half‐life (t_1/2=109.77 min), high cost and less availability of cyclotrone‐produced ¹⁸F make ^99mTc (t_1/2=6 h)‐based agents more desirable. With this aim, ^99mTc(CO)₃‐labeled iminodiacetic acid derivatives of 2‐ and 4‐nitroimidazole are investigated for their ability to target hypoxic tumors. A bifunctional chelating agent, N, N‐bis[(tert‐butoxycarbonyl)methyl]‐2‐bromoethylamine, was synthesized in the first step, which was then conjugated to the nitroimidazoles in the second step. The tert‐butyl ester derivatives formed were hydrolyzed to obtain the iminodiacetic acid derivatives of corresponding nitroimidazoles. The radiolabeling of the iminodiacetic acid derivatives with [^99mTc(CO)₃(H₂O)₃]⁺ core was carried out following a reported protocol. Biodistribution of the prepared complexes was carried out in Swiss mice bearing fibrosarcoma tumor. The 2‐nitroimidazole complex showed a steady retention in activity (～1.5% injected dose per gram [%ID/g]) in tumor throughout the period of study (3 h) indicating that it may be localized in the hypoxic cells. The 4‐nitroimidazole counterpart, however, showed an initial uptake of ～4.6%ID/g at 30 min post injection, which was then observed to wash out rapidly. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis of carbon‐14‐labeled isotopomer of 6‐(4‐methanesulfonylphenyl)‐5‐[4‐(2‐piperidin‐1‐yl‐ethoxy)phenoxy]‐naphthalen‐2‐ol HCL salt (LY2066948‐[14C] HCL salt)

Carbon‐14‐labeled 6‐(4‐methanesulfonylphenyl)‐5‐[4‐(2‐piperidin‐1‐yl‐ethoxy)phenoxy]naphthalen‐2‐ol, a novel selective estrogen receptor modulator (SERM) was synthesized. The key component, 6‐methoxy‐1‐tetralone‐[carbonyl‐¹⁴C], was synthesized from 3‐(3‐methoxyphenyl)‐propionic acid via an intra‐molecular Friedel–Crafts acylation of 4‐(3‐methoxyphenyl)butanoic acid‐[carboxy‐¹⁴C]. A palladium catalyzed alpha‐keto arylation of 6‐methoxy‐1‐tetralone with 4‐methanesulfonyl‐phenyl bromide, followed by a sequence of bromination, DDQ dehydrogenation, aryl Ullmann reaction, and demethylation with BBr₃ gave the desired product LY2066948‐[¹⁴C]. Copyright © 2007 John Wiley & Sons, Ltd.     

A novel and efficient asymmetric synthesis of carbon‐14 labeled (S,S)‐2,7‐di‐boc‐diamino[1,8‐14C2]suberic acid

Five hundred mCi of Potassium [¹⁴C]cyanide at a specific activity of 51 mCi/mmol was used to diastereoselectively introduce the carbon‐14 label into 1,6‐hexanedial via a thermodynamically controlled asymmetric Strecker reaction using (R)‐(‐)‐2‐phenylglycinol as the chiral auxiliary. The expected and predominant (R,S/S,R) diastereomer ( 2 ) was separated by preparative normal phase HPLC. The chiral auxiliary was removed by oxidation with lead tetraacetate and the resulting benzylimino nitrile exhaustively hydrolyzed in hydrochloric acid to give (S,S)‐2,7‐diamino[1,8‐¹⁴C₂]suberic acid ( 6 ). Subsequent acylation with di‐tert‐butyldicarbonate gave the title compound ( 1 ) with a radiochemical purity of 95.5%, a specific activity of 113 mCi/mmol, and an enantiomeric purity of 95.5% e.e. To our knowledge this is the first report of the asymmetric Strecker methodology being applied to the synthesis of a carbon‐14 labeled amino acid. Copyright © 2002 John Wiley & Sons, Ltd.     

Synthesis of 3H‐ and 2H4‐labelled versions of the hypoxia‐activated pre‐prodrug 2‐[(2‐bromoethyl)‐2,4‐dinitro‐6‐ [[[2‐(phosphonooxy)ethyl]amino]carbonyl]anilino]ethyl methanesulfonate (PR‐104)

³H‐ and ²H₄‐versions of the hypoxia‐activated pre‐prodrug PR‐104 [2‐[(2‐bromoethyl)‐2,4‐dinitro‐6‐[[[2‐(phosphonooxy)ethyl]amino]carbonyl]anilino]ethyl methanesulfonate], labelled in the ethylcarboxamide side chain, have been prepared, respectively, by [³H]NaBH₄ reduction of a precursor late stage aldehyde, and by late stage incorporation of deuterium with 2‐amino[1,1,2,2‐²H₄]ethanol. Copyright © 2007 John Wiley & Sons, Ltd.