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One-Pot Synthesis of Orthogonally Protected Enantiopure S-(Aminoalkyl)- cysteine Derivatives [] [a]

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One-Pot Synthesis of Orthogonally Protected Enantiopure S-(Aminoalkyl)- cysteine Derivatives [] [a]
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  FULL PAPER DOI: 10.1002/ejoc.200500464 One-Pot Synthesis of Orthogonally Protected Enantiopure  S  -(Aminoalkyl)-cysteine Derivatives [‡] Adele Bolognese, [a] Olga Fierro, [b] Daniela Guarino, [b] Luigi Longobardo,* [a,b] andRomualdo Caputo [a] Dedicated to the memory of Arno F. Spatola [‡‡] Keywords:  Thia diamino acids /  S  -Alkylation / Diversity / Cysteine /  β -Iodoamines The general synthesis of a new class of non-natural diaminoacids, 2-amino-3-[(2  -aminoalkyl)thio]propanoic acids or  S -(aminoalkyl)cysteines, is reported. Under the conditions de-vised, enantiopure  N  -Boc-protected  β -iodoamines, readilygenerated from proteinogenic  α -amino acids, are treatedwith  L -cysteine ethyl ester hydrochloride, using Cs 2 CO 3  as abase. The  S -alkylation products, obtained in high yields (96–98%) and without any detectable traces of accompanying Introduction Nowadays it is thoroughly recognized that natural pep-tides, despite the potential interest to exploit them as phar-maceutical lead compounds, are indeed poor drug candi-dates because of their low oral bioavailability, potential im-munogenicity and insufficient metabolic stability in vivo. [1] Recent efforts to ameliorate disadvantageous peptidecharacteristics and thus generate viable pharmaceuticaltherapies have focused on the creation of non-natural pep-tide mimics. These “peptidomimetics” can be based on anyoligomer that mimics peptide primary structure through theuse of amide bond isosteres and/or modification of thenative peptide backbone, including chain extension or het-eroatom incorporation. [2] Peptidomimetic oligomers areoften protease-resistant and may have reduced immunoge-nicity and improved bioavailability relative to peptide ana-logues.In addition to primary structural mimicry, a select subsetof the sequence-specific peptidomimetic oligomers, the so-called “foldamers”, [3] exhibits well-defined secondary struc- [‡] Chiral Aminoalkyl Cation Equivalents, 1[a] Dipartimento di Chimica Organica e Biochimica, Università diNapoli Federico II4, via Cynthia, 80126 Napoli, ItalyFax: +39-081-674102E-mail: luilongo@unina.it[b] CNR-Istituto di Scienze dell’Alimentazione 52,Via Roma, 83100 Avellino, Italy[‡‡]A pioneer of pseudopeptide chemistrySupporting information for this article is available on theWWW under http://www.eurjoc.org or from the author. Eur. J. Org. Chem.  2006 , 169–173 © 2006 Wiley-VCH Verlag GmbH & Co. KgaA, Weinheim  169 byproducts, are hydrolysed to yield the free carboxyl group.An orthogonal protection is then introduced on the freeamino group by treatment with Fmoc-OSu under standardconditions. The inclusion of one of these orthogonally pro-tected diamino acids in a solid-phase growing pentapeptideis also reported.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,Germany, 2006) tural elements such as helices, turns and small sheetlikestructures. When peptide bioactivity is contingent upon aprecise 3-D structure, the capacity of a biomimetic oligomerto fold can be indispensable. Examples of simple peptidomi-metics include azapeptides, oligocarbamates and oligoureas,and common examples of foldamers include  β -peptides,  γ -peptides, oligo(phenylene ethynylene)s, vinylogous sulfono-peptides and poly- N  -substituted glycines (peptoids). [2–5] However, the main road to generate peptide diversity stillresides on the preparation of non-natural amino acids andtheir incorporation in specific (bioactive) peptide sequences.In this paper, we report the enantioselective synthesis of new thia diamino acids  1  that represent a new class of non-natural amino acids resembling natural lanthionine ( 2 ) [6] one of whose carboxyl groups has been replaced by the sidechain (R in  1 ) of a proteinogenic  α -amino acid.To the best of our knowledge, the sole literature pre-cedent for these compounds is the so-called “thialysine”(“Tlys”) (R = H in  1 ), known for its interesting cytotoxicproperties towards human acute leukaemia Jurkat T cells. [7] Thialysine, whose preparation dates back to nearly half acentury ago, [8,9] should formally represent the parent com-pound of this class of thia diamino acids.In view of the biological properties that could be ex-pected for such compounds and also their usefulness in in-  A. Bolognese, O. Fierro, D. Guarino, L. Longobardo, R. Caputo FULL PAPER troducing elements of diversity if incorporated in syntheticpeptide molecules, we have realized a simple and efficientsynthesis that leads to orthogonally protected thia diaminoacids  1 , by  S  -alkylation of   C  -protected   -cysteine with dif-ferent  N  -Boc- β -iodoamines  5 , in their turn prepared as de-picted in Scheme 1. Scheme 1. Conversion of   N  -Boc- α -amino acids into  N  -Boc- β -iodoamines. i) THF, NMM, MeOCOCl, then NaBH 4  in H 2 O; ii)TPP-I 2 , ImH (imidazole), DMC, reflux, 1 h. In fact, the reaction is known, and several reports of   S  -alkylation of   N  -Boc- β -iodoamines by miscellaneous mer-captans can be found in the current literature. [10–14] The ra-tionale of their use in the synthesis of thia diamino acids  1 lies in the opportunity to access a series of synthetic build-ing blocks, chiral aminoalkyl cation equivalents that carrythrough the chirality of their parent proteinogenic  α -aminoacids. Results and Discussion The enantiopure  N  -Boc- β -iodoamines  5a  –  c  were readilyprepared in excellent yields from the corresponding natural α -amino acids  3a  –  c , as depicted in Scheme 1. The  N  -Boc-protected  β -amino alcohols  4a  –  c , coming from the re-duction of the  α -amino acids, were converted into the corre-sponding iodoamines by using a polymer-bound triarylpho-sphane/I 2  complex in dry dichloromethane (DCM) in thepresence of imidazole. [15] In consideration of the somewhathigh cost of such a triarylphosphane, we also carried outthe conversion by using the inexpensive triphenylphos-phane, with quite comparable results (see Exp. Sect.).Under our experimental conditions, the  N  -Boc- β -iodoamines  5a  –  c , generated from Phe, Leu and Pro, [16] respectively, were treated with   -cysteine ethyl ester hydro-chloride and Cs 2 CO 3 , in dry DMF under argon (Scheme 2).The  S  -alkylations proceeded in high yields (96–98%); nei-ther alkylation of the cysteinyl amino group nor traces of elimination products and/or aziridines from  5a  –  c  could bedetected by TLC or  1 H NMR of the crude alkylation prod-ucts  6a  –  c . www.eurjoc.org © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Eur. J. Org. Chem.  2006 , 169–173 170Scheme 2. Synthesis of orthogonally protected thia diamino acids.i) HCl ·  -Cys-OEt, Cs 2 CO 3 , DMF, room temp., 3 h; ii) aq. LiOH,MeOH, room temp., 2 h; iii) Fmoc-OSu, THF, Na 2 CO 3 , 0 °C, 1 h. The  S  -alkylation procedure may appear as a slight modi-fication of a method already reported for the synthesis of lanthionine derivatives. [17–19] Indeed, the resemblance isonly formal, since in our case the  S  -alkylation occurs on aniodide  β  to a carbamate function ( N  -Boc- β -iodoamines  5 )whereas the cysteine alkylation leading to lanthionines oc-curs on the  β -iodide of   N  -trityl-protected serine alkyl esterthat is naturally prone to undergo either hydrogen iodideelimination or aziridine ring closure.Following alkylation, the products  6a  –  c  were hydrolysedby LiOH in MeOH to the corresponding monoprotectedthia diamino acids  7a  –  c  that, without being isolated, weretreated with Fmoc-OSu under standard conditions. The fi-nal orthogonally protected thia diamino acids  8a  –  c  wereobtained in excellent (79–82%) overall yields (based on thestarting  N  -Boc- β -iodoamines) after purification by flashchromatography. Their regio- and stereochemical integritywas ascertained by TLC, RP-HPLC,  1 H and  13 C NMRspectroscopy, and MS analyses. DEPT and COSY  1 HNMR experiments also showed the absence of traces of other isomers.In order to avoid complicated systematic names in cur-rent laboratory practice, we propose for these new thia di-amino acids a nomenclature system in which each com-pound is given an acronym composed of “Cy” (from “Cys”)and the one-letter code denoting the  α -amino acid whoseside chain (R in formula  1 ) is present in it. A referee sug-gested using the one-letter code in lower case, to be more inconnection with the three-letter symbolism of amino acids.Accordingly, the three compounds we have synthesised andreported in this paper should be indicated as Fmoc-  -Cyf(Boc)-OH ( 8a ), Fmoc-  -Cyl(Boc)-OH ( 8b ) and Fmoc-  -Cyp(Boc)-OH ( 8c ).As an illustrative example of the versatility of our or-thogonally protected thia diamino acids, compound  8a  wasincluded in a growing pentapeptide on a PAC-PEG-PS (4-hydroxymethylphenoxyacetic acid polyethyleneglycol poly-  Orthogonally Protected Enantiopure  S  -(Aminoalkyl)cysteine Derivatives FULL PAPER styrene) support with a classical peptide coupling in DMF.HATU [2-(7-aza-1 H  -benzotriazole-1-yl)-1,1,3,3-tetrameth-yluronium hexafluorophosphate] and NMM ( N  -methyl-morpholine) were used as activating agents (Scheme 3). Scheme 3. Solid-phase synthesis of pentapeptide foldamer  9 . i) 2%DBU in DMF, Fmoc-Cyf(Boc)-OH, HATU, NMM, 1 h; ii) 2%DBU in DMF, Boc-Phe-OH, HATU, NMM, 30 min; iii) TFA,TES, TA, room temp., 2 h. In the final step, the product was cleaved from the poly-meric support, and the expected pentapeptide  9  was ob-tained. One single compound with the expected molecularweight could be detected by analytical RP-HPLC andMALDI-TOF MS analysis of the crude product. Conclusions We have proposed a synthesis of orthogonally protectedthia diamino acids that are ready to be inserted into solid-phase growing peptides to obtain foldamers.It is noteworthy that the same synthetic approach caneven be applied to the modification of native proteins (bio-conjugation), [20,21] considering that the most widely usedbioconjugation strategy exploits the latent nucleophilicityof the thiol side chain of cysteine. It might be interesting touse chiral  β -iodoamines as aminoalkyl cation equivalentsfor the  S  -alkylation of cysteines and the introduction of proteinogenic  α -amino acid moieties in native proteins.However, successful work is already in progress in ourlaboratory in preparing new orthogonally protected non-natural diamino acids such as  8a  –  c  with various hetero-atoms other than sulfur. Experimental Section General:  Melting points were measured with a Kofler apparatusand are uncorrected. Optical rotations were measured with a Jasco Eur. J. Org. Chem.  2006 , 169–173 © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org  1711010 polarimeter. RP-HPLC was carried out with a ShimadzuSCL-10 Avp system with a photodiode-array detector, using a bi-nary solvent system consisting of 0.01% TFA in H 2 O (solvent A)and 0.01% TFA in MeCN (solvent B); an analytical Vydac C 18 column was used (4.6 mm diameter with a flow rate of 1 mL/ min). 1 H and  13 C NMR spectra were recorded with Varian Inova 500and Bruker DRX-400 spectrometers: chemical shifts are in ppm (  δ  )and  J   coupling constants in Hz. Low-resolution MALDI-TOFmass spectra were obtained with a Voyager DE-PRO (PE-Biosys-tem), using 2,3-dihydroxybenzoic acid (DHB) as matrix. High-reso-lution ES mass spectra were obtained with a Micromass Q-TOFUltima TM API using Leu-Enk as standard and NaI for calibration.TLC was carried out on silica gel Merck 60 F254 plates (0.2 mmlayer thickness) and developed with ninhydrin (0.25% in MeOH)or visualized by UV. Column chromatography was performed onMerck Kieselgel 60 (70–230 mesh). Dry solvents were distilled im-mediately before use. Preparation of   N  -Boc- β -Iodoamines 5a–c. General Procedure:  To astirred suspension of polystyryl-diphenylphosphane (ca. 3 mmol/g,4 g, ca. 12 mmol) in dry DMC (70 mL), under argon, I 2  (3.62 g,12.7 mmol) and imidazole (1.70 g, 25 mmol) were added in se-quence. After 15 min, one of the  N  -Boc- β -aminols  4a  –  c  (5 mmol),dissolved in the same solvent (10 mL), was added in one portionto the heterogeneous reaction mixture, that was then refluxed for1 h (TLC monitoring, EtOAc/hexane, 15:85). After cooling, it wasdiluted with DMC (100 mL) and filtered through a glass septumfunnel to remove the polymeric material. The filtrate was washedwith aq. 10% Na 2 S 2 O 3  (2×50 mL) and finally with pure water,then concentrated in vacuo. The resulting  N  -Boc- β -iodoamines  5a  –  c , obtained in high yields (95–98%), were used directly in the nextstep. When, under the same experimental conditions, polystyryl-diphenylphosphane was replaced by soluble TPP (triphenylphos-phane) (1.64 g, 6.25 mmol), the final  N  -Boc- β -iodoamines neededfurther purification by flash chromatography (EtOAc/hexane, 7:3)and were obtained in somewhat lower yields (82–86%). tert  -Butyl [(1 S  )-1-Benzyl-2-iodoethyl)carbamate (5a):  96%; m.p.110–111 °C (from Et 2 O/hexane). [ α ] D20 = +18.3 ( c  = 1.3, CHCl 3 ){ref. [22] m.p. 118 °C; [ α ] D20 = +18.9 ( c  = 2.9)};  R f   (EtOAc/hexane,85:15) = 0.51.  1 H NMR (CDCl 3 , 500 MHz):  δ   = 1.42 (s, 9 H), 2.76(dd,  J   = 6.7 and 13.4 Hz, 1 H), 2.90 (dd,  J   = 5.0 and 13.4 Hz, 1H), 3.16 (dd,  J   = 3.3 and 10.0 Hz, 1 H), 3.38 (br. dd,  J   = 4.0 and10.0 Hz, 1 H), 3.45–3.70 (m, 1 H), 4.57–4.72 (m, 1 H), 7.20–7.40(m, 5 H) ppm. tert  -Butyl [(1 S  )-1-(Iodomethyl)-3-methylbutyl]carbamate (5b):  98%;m.p. 58 °C (from Et 2 O/hexane). [ α ] D20 = –31.6 ( c  = 1.5, CHCl 3 ){refs. [15,23] m.p. 55–57 °C; [ α ] D20 = –29.9 ( c  = 1.3)};  R f   (EtOAc/hex-ane, 85:15) = 0.56.  1 H NMR (CDCl 3 , 500 MHz):  δ   = 0.92 (d,  J   =6.7, 3 H), 0.93 (d,  J   = 6.7, 3 H), 1.30–1.37 (m, 2 H), 1.45 (s, 9 H),1.53–1.68 (m, 1 H), 3.28 (dd,  J   = 2.7 and 9.2 Hz, 1 H), 3.35–3.45(m, 1 H), 3.47 (dd,  J   = 3.7 and 9.2 Hz, 1 H), 4.52 (br. d,  J   = 6.0,1 H) ppm. tert  -Butyl [(1 S  )-2-Iodo-1-(pyrrolidin-1-yl)ethyl]carbamate (5c):  95%;m.p. 38–40 °C (from EtOAc/hexane). [ α ] D20 = –34.7 ( c  = 1.3, CHCl 3 ){ref. [24] m.p. 102–104 °C; [ α ] D20 = –32.8 ( c  = 1.5)};  R f   (EtOAc/hex-ane, 85:15) = 0.48.  1 H NMR (CDCl 3 , 500 MHz):  δ   = 1.46 (s, 9 H),1.81–1.83 (m, 1 H), 1.86–1.89 (m, 2 H), 2.03–2.05 (m, 1 H), 3.11– 3.15 (t,  J   = 9.5, 1 H), 3.34–3.37 (m, 2 H), 3.43–3.48 (t, 1 H), 3.82– 3.85 (m, 1 H) ppm. S  -Alkylations of Cysteine Ethyl Ester. General Procedure:  To astirred solution of one of the  β -iodoamines  5a  –  c  (0.33 mmol) and  -cysteine ethyl ester hydrochloride (62 mg, 0.33 mmol) in dryDMF (5 mL) and under argon, solid Cs 2 CO 3  (216 mg, 0. 66 mmol)  A. Bolognese, O. Fierro, D. Guarino, L. Longobardo, R. Caputo FULL PAPER was added in one portion. Stirring was continued in the dark atroom temperature for 3 h. Most of the solvent was carefully (  45 °C) evaporated in vacuo and the residue was suspended inEtOAc (50 mL) and shaken with H 2 O (3×20 mL). The organiclayer was then dried (Na 2 SO 4 ) and the solvents were evaporated invacuo. Ethyl (2 R )-2-Amino-3-{(2  S  )-2  -[( tert  -butyloxycarbonyl)amino]-3  -phenylpropylthio}propanoate, H-Cyf(Boc)-OEt (6a):  Oil, ca. 97%; R f   (DCM/MeOH, 95:5) = 0.23. Chromatographically pure sample: 1 H NMR (CDCl 3 , 400 MHz):  δ   = 1.24 (t, 3 H), 1.39 (s, 9 H), 2.62(d,  J   = 5.5, 2 H), 2.76–2.93 (m, 3 H), 2.9 (dd,  J   = 4.6 and 13.4 Hz,1 H), 3.60 (dd,  J   = 4.6 and 7.0 Hz, 1 H), 3.92–4.01 (m, 1 H), 4.15(q, 2 H), 5.03 (d,  J   = 8.4, 1 H), 7.16–7.28 (m, 5 H) ppm.  13 C NMR(CDCl 3 , 125 MHz):  δ   = 13.9, 28.1, 36.6, 37.7, 39.3, 51.2, 54.1, 61.0,79.0, 126.3, 128.2, 129.2, 137.4, 155.1, 173.7 ppm. C 19 H 30 N 2 O 4 S(382.5): calcd. C 59.66, H 7.91; found C 59.61, H 7.87 (speciallyprepared analytical sample). Ethyl (2 R )-2-Amino-3-{(2  S  )-2  -[( tert  -butoxycarbonyl)amino]-4  -methylpentylthio}propanoate, H-Cyl(Boc)-OEt (6b):  Oil, ca. 98%; R f   (DCM/MeOH, 95:5) = 0.21. Chromatographically pure sample: 1 H NMR (CDCl 3 , 400 MHz):  δ   = 0.82 (d, 6 H), 1.21 (t, 3 H), 1.31(t, 2 H), 1.41 (s, 9 H), 1.6 (m, 1 H), 2.61 (d,  J   = 5.5, 2 H), 2.75(dd,  J   = 7.0 and 13.4 Hz, 1 H), 2.79 (dd,  J   = 4.6 and 13.4 Hz, 1H), 3.51 (dd,  J   = 4.6 and 7.0 Hz, 1 H), 3.9 (m, 1 H), 4.18 (q, 2 H),4.9 (br. d, 1 H) ppm.  13 C NMR (CDCl 3 , 100 MHz):  δ   = 14.3, 22.3,23.2, 25.1, 28.6, 38.4, 39.1, 43.2, 48.6, 54.5, 61.3, 79.7, 155.6,174.1 ppm. C 16 H 32 N 2 O 4 S (348.5): calcd. C 55.14, H 9.26; found C55.20, H 9.27 (specially prepared analytical sample). Ethyl (2 R )-2-Amino-3-{[(2  S  )-1-( tert  -butoxycarbonyl)pyrrolidin-2  -yl]methylthio}propanoate, H-Cyp(Boc)-OEt (6c):  Oil, ca. 96%;  R f  (DCM/MeOH, 95:5) = 0.29. Chromatographically pure sample:  1 HNMR (CD 3 OD, 400 MHz):  δ   = 1.28 (t, 3 H), 1.45 (s, 9 H), 1.85– 1.97 (m, 4 H), 2.55–2.58 (m, 1 H), 2.84–2.88 (m, 3 H), 3.36–3.38(m, 2 H), 3.60–3.63 (m, 1 H), 3.87–3.89 (m, 1 H), 4.16–4.21 (q, 2H) ppm.  13 C NMR (CDCl 3 , 100 MHz):  δ   = 14.4, 22.9, 28.7, 30.2,36.5, 37.8, 47.2, 54.5, 57.1, 61.3, 79.8, 154.4, 174.1 ppm.C 15 H 28 N 2 O 4 S (332.5), calcd. C 54.19, H 8.49; found C 54.24, H8.43 (specially prepared analytical sample). Alkaline Hydrolysis of Crude 6a–c and Fmoc Protection of the CrudeDiamino Acids 7a–c. General Procedure:  To a stirred solution of one of the  S  -alkylated compounds  6a  –  c  (0.3 mmol) in MeOH(5 mL) aq. 1    LiOH (1 mL) was added in one portion. After 2 hof stirring at room temperature, MeOH was evaporated in vacuo.The residue was redissolved in THF (10 mL) and the solution co-oled to 0 °C. Fmoc-OSu (112 mg, 0.33 mmol) was then added por-tionwise over 1 h, maintaining an alkaline pH (ca. 10) by dropwiseaddition of 10% aq. Na 2 CO 3 , then by addition of excess solidNa 2 CO 3 . THF was evaporated in vacuo and the crude reactionproduct was dissolved in EtOAc (75 mL) and worked up as usual,under acidic conditions (aq. 0.1    HCl). The final  N  -Fmoc, N   -Bocthia diamino acids  8a  –  c  were purified by flash chromatography,using a 0–5% MeOH gradient in DCM. (2 R )-3-[(2  S)-2  -( tert  -Butyloxycarbonyl)amino-3  -phenylpropylthio]-2-[(fluorenylmethoxycarbonyl)amino]propanoic Acid, Fmoc-Cyf(Boc)-OH (8a):  80% (overall yield from  5a );  R f   (DCM/MeOH,9:1) = 0.47. Analytical sample, crystallized from hot absoluteEtOH: M.p. 135–136 °C. [ α ] D20 = –20.0 ( c  = 1.0, MeOH).  1 H NMR(CD 3 OD, 500 MHz):  δ   = 1.40 (s, 9 H), 2.62–2.67 (m, 3 H), 2.84– 2.90 (m, 2 H), 3.04–3.06 (dd,  J   = 4.5 Hz, 1 H), 3.86–3.87 (m, 1 H),4.22–4.27 (t,  J   = 7.7, 1 H), 4.31–4.37 (m, 3 H), 7.13–7.20 (m, 5 H),7.27–7.31 (t,  J   = 7.5 Hz, 2 H), 7.35–7.39 (t, 2 H), 7.67–7.69 (d,  J  = 7.4 Hz, 2 H), 7.77–7.79 (d, 2 H) ppm.  13 C NMR (CD 3 OD, www.eurjoc.org © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Eur. J. Org. Chem.  2006 , 169–173 172125 MHz):  δ   = 29.9, 36.8, 39.5, 42.1, 54.6, 56.6, 59.5, 69.3, 81.2,122.0, 127.5, 128.4, 129.3, 129.9, 130.4, 131.5, 140.9, 143.7, 146.4,170.2 ppm. MS-MALDI-TOF (C 32 H 36 N 2 O 6 S):  m / z  = 599.14 [M +Na + ], 615.18 [M + K + ], 477.30 [M – Boc]. HR ES-MS (EI): calcd.577.2765 [MH + ], found 577.2789. (2 R )-3-[(2  S  )-2  -( tert  -Butyloxycarbonyl)amino-4  -methylpentylthio]-2-[(fluorenylmethoxycarbonyl)amino]propanoic Acid, Fmoc-Cyl-(Boc)-OH (8b):  82% (overall yield from  5b );  R f   (DCM/MeOH, 9:1)= 0.37. Analytical sample, crystallized from hot absolute EtOH:M.p. 130–133 °C. [ α ] D20 = –29.2 ( c  = 0.6, MeOH).  1 H NMR(CD 3 OD, 500 MHz):  δ   = 0.88 (d,  J   = 5.8 Hz, 6 H), 1.28–1.36 (m,2 H), 1.42 (s, 9 H), 1.62–1.65 (m, 1 H), 2.61–2.64 (m, 2 H), 2.91– 2.94 (m, 1 H), 3.05–3.08 (m, 1 H), 3.66–3.70 (m, 1 H), 4.23–4.33(m, 4 H), 7.29–7.32 (t,  J   = 7.5 Hz, 2 H), 7.36–7.40 (t, 2 H), 7.69– 7.70 (d,  J   = 7.4 Hz, 2 H), 7.78–7.80 (d, 2 H) ppm.  13 C NMR(CD 3 OD, 125 MHz):  δ   = 19.5, 29.9, 36.8, 39.5, 42.1, 54.6, 56.6,59.5, 69.3, 81.1, 122.0, 127.5, 129.3, 129.9, 130.4, 131.5 140.9,143.7, 146.4, 159.0, 159.6, 160.5, 171.4 ppm. MS-MALDI-TOF(C 29 H 38 N 2 O 6 S):  m / z  = 565.32 [M + Na + ], 581.33 [M + K + ], 443.25[M – Boc]. HR ES-MS (EI): calcd. 543.2940 [MH + ], found543.2965. (2 R )-3-{[(2  S  )-1-( tert  -Butyloxycarbonyl)pyrrolidin-2  -ylmethyl]-thio}-2-[(fluorenylmethoxycarbonyl)amino]propanoic Acid, Fmoc-Cyp(Boc)-OH (8c):  79% (overall yield from  5c );  R f   (DCM/MeOH,9:1) = 0.45. Analytical sample, crystallized from hot absoluteEtOH: M.p. 130–133 °C. [ α ] D20 = –25.2 ( c  = 0.6, MeOH).  1 H NMR(CD 3 OD, 500 MHz):  δ   = 1.36 (s, 9 H), 1.67–1.69 (m, 1 H), 1.80– 1.85 (m, 3 H), 2.49–2.57 (m, 1 H), 2.76–2.81 (m, 3 H), 2.99–3.01(m, 2 H), 3.07–3.10 (m, 1 H), 3.80–3.82 (m, 2 H), 4.13–4.16 (t,  J   =7.7 Hz, 1 H), 4.20–4.26 (m, 3 H), 7.20–7.22 (t,  J   = 7.5 Hz, 2 H),7.26–7.28 (t, 2 H), 7.57–7.59 (d,  J   = 7.4 Hz, 2 H), 7.68–7.70 (d,  J  = 7.4 Hz, 2 H) ppm.  13 C NMR (CD 3 OD, 125 MHz):  δ   = 22.1, 28.1,29.1, 33.6, 35.6, 46.3, 52.4, 57.1, 65.7, 79.8, 120.1 125.3, 127.1,127.6, 140.7, 143.8, 153.9, 157.2, 172.3 ppm. MS-MALDI-TOF(C 28 H 34 N 2 O 6 S):  m / z  = 549.6 [M + Na + ], 565.7 [M + K + ], 426.5[M – Boc]. HR ES-MS (EI): calcd. 527.2731 [MH + ], found527.2704. Solid-Phase Synthesis of H-Phe-Cyf-Asp-Lys-Gln-OH (9):  In amanual peptide synthesizer Fmoc-Asp(O t Bu)-Lys(Boc)-Gln(Trt)-PAC-PEG-PS was grown up starting from Fmoc-Gln(Trt)-PAC-PEG-PS (118 mg, 0.16 mmol/g, 0.019 mmol). The Fmoc group wasremoved using 2% DBU (1,8-diazobicyclo[5.4.0]undec-7-ene) inDMF (3 mL) for 30 min. The orthogonally protected thia diaminoacid  8a  (43 mg, 0.076 mmol) was then added together with HATU(29 mg, 0.076 mmol) and NMM (8  µ L) in DMF (3 mL). The coup-ling proceeded for 1 h. The resin was washed with DMF (3×5 mL)and DCM (3×5 mL) and the protecting Fmoc group was againremoved with 2% DBU in DMF (3 mL) for 30 min. The last coup-ling was performed by using Boc-Phe-OH (20 mg, 0.076 mmol),HATU (29 mg, 0.076 mmol) and NMM (8  µ L) in DMF (3 mL).The product was eventually washed with DMF, DCM, and Et 2 O,dried and cleaved from the polymeric support using a mixture of TFA (1.9 mL), TES (triethylsilane) (60  µ L) and TA (thioanisole)(60  µ L) at room temperature for 2 h. After precipitation in dryEt 2 O, it was washed 3 times with the same solvent. Under analyti-cal RP-HPLC conditions (solvents A/B, 95:5 over 5 min, then from95:5 to 8:2 over 10 min, finally from 8:2 to 1:1 over 10 min) a singlepeak could be detected at  t  = 12.6 min (220 nm). MS-MALDI-TOF (C 36 H 52 N 8 O 9 S):  m / z  (%) = 773.24 (100) [M], 795.21 (50) [M+ Na + ], 811.21 (10) [M + K + ], 755.23 (30) [M – H 2 O]. HR ES-MS(EI): calcd. 773.3746 [MH + ], found 773.3759.  Orthogonally Protected Enantiopure  S  -(Aminoalkyl)cysteine Derivatives FULL PAPER Supporting Information:  Supporting information for this article, in-cluding full characterisation ( 1 H NMR,  13 C NMR, MALDI-TOFand HR MS) of compounds  6a  –  c  throughout  9  is available (see thefootnote on the first page of this paper). Acknowledgments The authors thank Dr. Pasquale Festa and Dr. Michele Manfrafor assistance in NMR spectroscopy and Dr. Luca Picariello forMALDI-TOF MS and ES-MS analyses.[1] P. W. Latham,  Nat. Biotechnol.  1999 ,  17  , 755–757.[2] A. F. Spatola, “Peptide Backbone Modifications”, in  Chemis-try and Biochemistry of Amino Acids, Peptides, and Proteins ,vol. VII (Ed.: B. Weinstein), Marcel Dekker, New York,  1983 ,pp. 267–357.[3] S. H. Gellman,  Acc. Chem. Res.  1998 ,  31 , 173–180.[4] K. Kirshenbaum, R. N. Zuckermann, K. A. Dill,  Curr. Opin.Struct. Biol.  1999 ,  9 , 530–535.[5] A. E. Barron, R. N. 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Odagaki, S. Oishi, H.Tamamura, N. Hamanaka, N. Fujii,  J. Org. Chem.  2002 ,  67  ,6152–6161.[15] R. Caputo, E. Cassano, L. Longobardo, G. Palumbo,  Tetrahe-dron Lett.  1995 ,  36  , 167–168.[16]  N  -Boc- β -iodoamine  5c  from proline is known (ref. [24] ), and itsmelting point is reported to be 102–104 °C. Unfortunately, inour hands, such a compound, obtained through three indepen-dent preparations, constantly turned out to be low-melting: thehighest measured melting point was 38–40 °C. The rotations,however, seemed to fit quite well. Apparently, the value of themelting point previously reported for  5c  should be revised.[17] C. Dugave, A. Menez,  J. Org. Chem.  1996 ,  61 , 6067–6070.[18] C. Dugave, A. Menez,  Tetrahedron: Asymmetry  1997 ,  8 , 1453– 1465.[19] V. Swali, M. Matteucci, R. Elliot, M. Bradleya,  Tetrahedron 2002 ,  58 , 9101–9109.[20] B. G. Davis,  Curr. Opin. Biotechnol.  2003 ,  14 , 379–386.[21] D. F. Qi, C. M. Tann, D. Haring, M. D. Distefano,  Chem. Rev. 2001 ,  101 , 3081–3112.[22] R. Duddu, M. Eckhardt, M. Furlong, H. P. Knoess, S. Berger,P. Knochel,  Tetrahedron  1994 ,  50 , 2415–2432.[23] M. Jost, J. C. Greie, N. Stemmer, S. D. Wilking, K. Altendorf,N. Sewald,  Angew. Chem. Int. Ed.  2002 ,  41 , 4267–4269.[24] S. H. Park, H. J. Kang, S. Ko, S. Park, S. Chang,  Tetrahedron:Asymmetry  2001 ,  12 , 2621–2624.Received: June 24, 2005Published Online: November 7, 2005
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