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temperature overnight. The resin is filtered, washed with DMF and DCM, and dried under vacuum overnight, as before, to give resin 17. For the Suzuki cleavage reaction, a portion of the dry resin 17 (0.20 g, 0.07 mmol) is mixed with Pd(dppf)Cl2 (7.2 mg), an arylboronic acid (0.25 mmol), TEA (0.1 ml, 0.60 mmol), and DMF (2.0 ml) in a glass vial
under nitrogen. The mixture is then shaken at 90 overnight. The polymer beads are next filtered and washed several times with Et2O and the combined organic phase is washed with aqueous 2% sodium carbonate and water and then evaporated to dryness. The crude products are purified by preparative TLC (or other suitable methods) to give the desired products 18 in 65–90% yields, with >98% purity as determined by HPLC. General Procedure for Cleavage of Phenols by a Reductive Elimination Reaction: Preparation of Compounds 20 and 22 To dried resins 19 and 21 (0.1 g resin, approximately 0.04 mmol with respect to the loading of the phenol) are added Pd(OAc)2 (8.0 mg), 1,3-bis (diphenyl-phosphino)propane (dppp, 17.0 mg), DMF (1.4 ml), and a mix
ture of HCO2H (0.2 ml) and TEA (0.8 ml). The mixture is shaken at 85 for 2 h, and then the resin is filtered and washed several times with diethyl ether. The combined organic phase is washed with aqueous sodium carbonate solution then water and evaporated to dryness. The residue obtained is dissolved in diethyl ether and eluted through a short column of alumina to remove any remaining inorganic residues. The crude products are purified by preparative TLC (or other suitable methods) to give the desired products 20 and 22 in >95% purity.

[10] Unnatural Diamino Acid Derivatives as Scaffolds for Creating Diversity and as Linkers for Simplifying Screening in Chemical Libraries By Robert Pascal, Re´gine Sola, and Patrick Jouin Introduction

The introduction of conformational restrictions into flexible active molecules is a well-known strategy for trying to increase their potency and/or selectivity toward their biological targets.1 Several methods have been used for constraining flexible molecules. Cyclic derivatives of linear peptides or peptidomimetics can thus be prepared by reactions involving side-chain

METHODS IN ENZYMOLOGY, VOL. 369

Copyright 2003, Elsevier Inc. All rights reserved. 0076-6879/03 $35.00

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NH2 H 2N

R

H 2N

COOH 1

H 2N

COOH 2

H2N (CH2)n HN

COOH

3 (n = 2,3)

Fig. 1. Unnatural aliphatic diamino acids.

functional groups and/or C- or N-termini.2 For this purpose, lactam bridges linking Lys and Asp residues have been introduced in peptides.3 However, selecting linear oligomers prior to introducing conformational restrictions is not essential and combinatorial chemistry has indeed been successful in the direct production of lead compounds from chemical libraries.4 A powerful method is to build libraries starting from suitable scaffolds that display several pendant functional groups to introduce diversity.1,5–7 In this context, amino acids bearing an extra amino functionality are potentially very attractive either for peptide cyclization or as central scaffold structures displaying three points of diversity. A major advantage of carboxyl and amino groups is their compatibility with standard protocols of peptide synthesis. Moreover, hydrophilic amide linkages present in the products are likely to increase their bioavailability as potential drugs. Finally, constructions based on amide bonds are usually chemically stable and their stability toward proteases is likely to be increased if unnatural diamino acids (Fig. 1) are involved. In spite of these useful features, few unnatural diamino acids with appropriate protecting groups have been reported and still fewer are commercially available. A survey of such structures is presented here and some of their possible applications in combinatorial chemistry are mentioned or illustrated by methodological developments carried out in our research group: the use of derivatives of benzoic acid as scaffolds for creating diversity and a procedure for handling a linker for solid-phase synthesis derived from l-2,3-diaminopropionic acid 1

E. M. Gordon, R. W. Barrett, W. J. Dower, S. P. A. Fodor, and M. A. Gallop, J. Med. Chem. 37, 1385 (1994). 2 J. N. Lambert, J. P. Mitchell, and K. D. Roberts, J. Chem. Soc. Perkin Trans. 1, 471 (2001). 3 W. Zhang and J. W. Taylor, Tetrahedron Lett. 37, 2173 (1996) and references cited therein. 4 A. Golebiowski, S. R. Klopfenstein, and D. E. Portlock, Curr. Opin. Chem. Biol. 5, 273 (2001). 5 A. J. Souers and J. A. Ellman, Tetrahedron 57, 7431 (2001). 6 J. A. Ellman, Acc. Chem. Res. 29, 132 (1996). 7 M. Royo, M. del Fresno, A. Frieden, W. Van Den Nest, M. Sanseverino, J. Alsina, S. A. Kates, G. Barany, and F. Albericio, React. Funct. Polym. 41, 103 (1999).

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4 H2N

NH2

H2N

NH2

COOH

COOH

COOH

H2N

H2N

*

6

*

* COOH N H 7

NH2

H2N

5

COOH * COOH

HN NH2

N H 8

9

H N

HN

COOH

COOH NH 10

N H 11

Fig. 2. Unnatural cyclic diamino acids.

(1) (Dpr) and allowing the mild release of molecules in media that can be readily made compatible with biological assays. Two lower homologues of lysine, Dpr3,8–10 and l-2,4-diaminobutyric acid (2) (Dab), have often been used to introduce conformational restrictions on peptides by lactam bridges. The Dpr to Asp linkage can also be used as a stable surrogate of disulfide bonds for stabilizing loops.11,12 Except for small cyclic systems, the residual conformational flexibility is not likely to provide an appropriate rigidity to structures based on Dpr or Dab residues. A similar finding can be applied to the backbone-to-backbone cyclization strategy based on the use of N-aminoalkyl amino acid residues 3.13 In our opinion, better rigidity may be expected from cyclic building blocks (Fig. 2). Compounds 4–6 containing three- or four-membered rings have been prepared under conveniently protected forms.14,15 They have 8

J. Rizo, S. C. Koerber, R. J. Bienstock, J. Rivier, A. T. Hagler, and L. M. Gierasch, J. Am. Chem. Soc. 114, 2852 (1992). 9 C. H. Hassall, R. G. Tyson, and K. K. Chexal, J. Chem. Soc. Perkin Trans. 1, 2010 (1976). 10 P. Wipf and H.-Y. Kim, Tetrahedron Lett. 33, 4275 (1992). 11 D. Limal, J.-P. Briand, P. Dalbon, and M. Jolivet, J. Peptide Res. 52, 121 (1998). 12 C. Mendre, R. Pascal, and B. Calas, Tetrahedron Lett. 35, 5429 (1994). 13 B. Mu¨ ller, D. Besser, P. Kleinwa¨ chter, O. Arad, and S. Reissmann, J. Peptide Res. 54, 383 (1999). 14 T. Wakamiya, Y. Oda, H. Fujita, and T. Shiba, Tetrahedron Lett. 27, 2143 (1986). 15 E. Gershonov, R. Granoth, E. Tzehoval, Y. Gaoni, and M. Fridkin, J. Med. Chem. 39, 4833 (1996).

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been mainly used as constrained analogues of lysine or ornithine or as intermediates in the preparation of analogues of arginine. Synthetic routes giving access to building blocks derived from all the stereoisomers of 4-aminoproline (7) and its homologue 8 have also been devised.16–18 Residue 9 has been used to promote helix formation in peptides.19 Carboxylic acids 10 and 11, derived from piperazine and imidazolidine, respectively, have been synthesized with orthogonal amino-protecting groups.20–22 The structure of residue 10 is also found in several -turn mimetics.5 Although many residues in Fig. 2 might be used as scaffolds for combinatorial synthesis, it is interesting to point out that only cis-aminoprolines 7 have been considered for such applications.7 Several other protected diamino acids involve aromatic rings (Fig. 3).23–27 Their structures may also be suitable as scaffolds for building synthetic libraries, but this application has been proposed only for compounds 12, 15, and 16.24,25 Whereas most of the structures displayed in Fig. 2 require stereoselective syntheses, the preparation of diamino acids 12–17 is facilitated by the absence of an asymmetric center. One of the most important limitations in the use of these diamino acids is the need of an additional orthogonal protection for the amino groups. Using the t-butoxycarbonyl/benzyl (Boc/Bzl) strategy of solid-phase peptide synthesis, this additional orthogonality can be easily provided by a base-labile protecting group such as the 9-fluorenylmethyloxycarbonyl 16

T. R. Webb and C. Eigenbrot, J. Org. Chem. 56, 3009 (1991). Z. Zhang, A. Van Aerschot, C. Hendrix, R. Busson, F. David, P. Sandra, and P. Herdewijn, Tetrahedron 56, 2513 (2000). 18 M. Tamaki, G. Han, and V. J. Hruby, J. Org. Chem. 66, 1038 (2001). 19 C. L. Wysong, T. S. Yokum, G. A. Morales, R. L. Gundry, M. L. McLaughlin, and R. P. Hammer, J. Org. Chem. 61, 7650 (1996). 20 B. D. Dorsey, R. B. Levin, S. L. McDaniel, J. P. Vacca, J. P. Guare, P. L. Darke, J. A. Zugay, E. A. Emini, W. A. Schleif, J. C. Quintero, J. H. Lin, I.-W. Chen, M. K. Holloway, P. M. D. Fitzgerald, M. G. Axel, D. Ostovic, P. S. Anderson, and J. R. Huff, J. Med. Chem. 37, 3443 (1994). 21 A. M. Warshawsky, M. V. Patel, and T.-M. Chen, J. Org. Chem. 62, 6439 (1997). 22 L. Rene´ , L. Yaouancq, and B. Badet, Tetrahedron Lett. 39, 2569 (1998). 23 R. M. Keenan, J. F. Callahan, J. M. Samanen, W. E. Bondinell, R. R. Calvo, L. Chen, C. DeBrosse, D. S. Eggleston, R. C. Haltiwanger, S. M. Hwang, D. R. Jakas, T. W. Ku, W. H. Miller, K. A. Newlander, A. Nichols, M. F. Parker, L. S. Southhall, I. Uzinskas, J. A. Vasko-Moser, J. W. Venslavsky, A. S. Wong, and W. F. Huffman, J. Med. Chem. 42, 545 (1999). 24 B. R. Neustadt, E. M. Smith, T. Nechuta, and Y. Zhang, Tetrahedron Lett. 39, 5317 (1998). 25 R. Pascal, R. Sola, F. Labe´ gue`re, and P. Jouin, Eur. J. Org. Chem. 3755 (2000). 26 M. H. Gelb and R. H. Abeles, J. Med. Chem. 29, 585 (1986). 27 V. Santagada, F. Fiorino, B. Severino, S. Salvadori, L. H. Lazarus, S. D. Bryant, and G. Caliendo, Tetrahedron Lett. 42, 3507 (2001). 17

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Fmoc NH

NH Boc

NH2 COOH

Boc NH COOH

Z N H

COOH

H2N 12

13

Boc N H H2N

COOH 15

Fmoc N H H2N

14

COOH

Boc N H Fmoc HN

16

COOH 17

COOH N

H2N

Fmoc

18

Fig. 3. Unnatural arylamino amino acid building blocks.

(Fmoc) group. Using the Fmoc/t-butyl strategy, three main orthogonal classes of protecting groups are available and have been recently reviewed.28 The first class consists of highly acid-labile groups such as 2-(4-biphenyl)isopropoxycarbonyl (Bpoc), trityl (Trt), or derivatives, and , -dimethyl -3,5-dimethoxybenzyloxycarbonyl (Ddz), which can be removed in the presence of the t-butyl group with practically complete selectivity. The second one involves groups removed by hydrazine such as the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) group or preferentially its isovaleryl analogue, Ddiv, which may avoid the intramolecular migration observed with the Dde group.29 The third one involves the palladium-labile allyloxycarbonyl group (Alloc). However, in the case of the aromatic protected scaffolds 12, 15, and 16, the protection of the arylamino group was shown to be useless because it can be replaced by the choice of selective coupling conditions.24,25 Our interest in diamino acid building blocks or scaffolds is connected with the studies of a new type of safety-catch linkers based on a diamino acid residue that we have carried out.30–32 Safety-catch linkers33 for 28

F. Albericio, Biopolymers 55, 123 (2000). S. R. Chhabra, B. Hothi, D. J. Evans, P. D. White, B. W. Bycroft, and W. C. Chan, Tetrahedron Lett. 39, 1603 (1998). 30 R. Sola, P. Saguer, M.-L. David, and R. Pascal, J. Chem. Soc. Chem. Commun. 1786 (1993). 29

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H2N H2N

COOH

i, ii, iii

H2N

COOH

iv

15 or 16

AmAbz

Fig. 4. Preparation of the AmAbz scaffold and of its protected derivatives 15 and 16. (i) MeOCOCl, Na2SO4, dioxane; (ii) N-hydroxymethylphthalimide, 96% H2SO4–H2O (9:1, v/v); (iii) NaOH, H2O; (iv) Boc2O or Fmoc–OSu, NaOH, dioxane-H2O.

solid-phase synthesis are designed to be cleaved by performing two different reactions with the advantage of providing an increased stability during the synthesis. Indeed, the Dpr(Phoc) linker30–32 (Phoc ¼ phenyloxycarbonyl) is incorporated by formation of amide bonds as stable as any peptide bond, but it can be activated as a cyclic N-acylurea and, thereby, made sensitive to nucleophiles at the end of the synthesis. It is well suited for the synthesis of peptides or peptidomimetics on hydrophilic supports and it is compatible with Boc and Fmoc strategies. Moreover, due to the stability of this linker in strongly acidic media, side-chain-protecting groups can be removed in an independent step preceding the cleavage in weakly alkaline aqueous solution. This linker could then become a powerful tool for the preparation of libraries of peptides or peptidomimetics free of deprotection contaminants and suitable for direct biological assays after addition of an appropriate buffer. The Dpr(Phoc) linker is therefore fully compatible with the high-throughput screening of libraries synthesized via solid phase, which requires ready purification procedures. Indications that related aromatic structures might improve the cleavage rate have also been reported.34 Methodology for the Use of the Protected Aromatic Scaffold 16

4-Amino-3-(aminomethyl)benzoic acid (AmAbz) can be easily prepared in three steps by amidomethylation of aminobenzoic acid (Fig. 4).25 Then the benzylamino group can be selectively protected by reaction with mild reagents such as Boc2O or Fmoc–OSu capable of discriminating between the two amino groups to give the building blocks AmAbz(Boc) (15) and AmAbz(Fmoc) (16), respectively (by convention, the 4-amino group is defined here as the main chain and the 3-aminomethyl group as 31

R. Sola, J. Me´ ry, and R. Pascal, Tetrahedron Lett. 37, 9195 (1996). R. Pascal and R. Sola, Tetrahedron Lett. 38, 4549 (1997). 33 M. Patek and M. Lebl, Biopolymers 47, 353 (1998). 34 R. Pascal, D. Chauvey, and R. Sola, Tetrahedron Lett. 35, 6291 (1994). 32

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Fig. 5. Solid-phase synthesis of the AmAbz scaffold-containing heptapeptide 19. (i) Piperidine–DMF (1:4);(ii) amino acid building block, BOP/HOBt/DIEA, DMF;(iii) FmocPhe, DIC, CH2Cl2; (iv) TFA, H2O, TIS (95:2.5:2.5, v/v/v).

the side chain for building abbreviations). The potential application of this scaffold to the preparation of a library containing the 6.4  107 heptapeptides obtained by combination of the 20 natural amino acids is illustrated by the solid-phase synthesis of the branched heptapeptide 19 (Fig. 5). Aminomethylpolystyrene resin (0.56 mmol/g) is derivatized with the Rink amide linker and the two residues (Gly and Val) at the C-terminus can be introduced by standard Fmoc-based solid-phase methods of peptide synthesis. At that time, AmAbz(Fmoc), which is now commercially available, can be introduced using BOP* activation in the presence of HOBt. With *

Abbreviations: BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; DCC, N,N0 -dicyclohexylcarbodiimide; DIC, N,N0 -diisopropylcarbodiimide; DME, dimethoxyethane; DMF, N,N-dimethylformamide; DIEA, N-ethyldiisopropylamine; Et2O, diethyl ether; HOBt, 1-hydroxybenzotriazole; HOSu, N-hydroxysuccinimide; t-BuOMe, tert-butylmethylether; TFA, trifluoroacetic acid; TIS, triisopropylsilane.

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this acylation method, involving hydroxybenzotriazole (HOBt) active esters, the free arylamino group is unaffected.25 The next residues can be selectively attached to the benzylamino group by using the same method. Then, more powerful conditions are required to acylate the arylamino group. Although PyBroP with diisopropylethylamine (DIEA) in CH2Cl2 has been reported to be efficient,24 we have preferred a coupling procedure involving diisopropylcarbodiimide (DIC) activation in CH2Cl2 because basic conditions are likely to promote the racemization of the activated amino acid. As a matter of fact, the arylamino group is not protonated in the medium owing to its low basicity and can thus be acylated even in the absence of base. The next residues can be introduced using standard coupling conditions. The side-chain-protecting groups can be removed and the product released by acidolysis. Methodology for the Use of Dpr(Phoc) Linker

The attachment of the Dpr(Phoc) residue to the support can be made, as previously reported,30–32 by coupling the Boc-Dpr(Phoc) building block. It is worth mentioning that this building block could not be obtained as a crystalline solid, a practical consideration to keep in mind when using this linker. An improvement based on the preparation of the crystalline activated ester Boc-Dpr(Phoc)–OSu is presented here as a convenient alternative route as well as the procedures for applying it to Boc or Fmoc methods of solid-phase synthesis (Fig. 6). Before the cleavage by mild alkaline hydrolysis into carboxylic acid, the stable C-terminal amide linkage must be converted into the labile acylurea via an intramolecular reaction induced by the breakdown of the phenyl carbamate moiety under mild alkaline conditions. At this activation stage, high selectivity is needed. To prevent any side reaction of the preceding amide group at this stage, the linker must be attached to solid supports bearing secondary amino groups. Thus Tentagel S-NH2 resin can be modified with Boc-Sar (Sar ¼ N-methylglycine). After the deprotection step, Boc-Dpr(Phoc)–OSu is reacted with the resin in the presence of HOBt and DIEA. The reaction generally proceeds to completion within 15–20 h as indicated by the chloranil test.35 The resin can then be used for solid-phase synthesis. Using Fmoc strategy, the linker must be cyclized into the Imc (Imc ¼ 2-oxoimidazoline-5-carboxylic acid residue) form, which is resistant to piperidine treatment.31 This operation can be better carried out after the attachment of the C-terminal residue except for sequences that are prone to diketopiperazine formation, which then

35

T. Vojkovsky, Peptide Res. 8, 236 (1995).

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Fig. 6. Use of Dpr(Phoc) linker in Boc (left) or Fmoc (right) chemistries. PG, Boc or Fmoc; scPGs, side-chain-protecting groups.

would require special treatment.31 After peptide chain elongation, the sidechain-protecting groups can be removed with the usual reagents for Boc or Fmoc strategies prior to the alkaline cleavage of the linker; purification steps are not essential when using this procedure since the deprotection

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contaminants are removed by simply washing the resin. Finally, crude products of acceptable purity can generally be released in solution from the support by a 1–3 h treatment with 0.01 N NaOH in water or 2-propanol–water (7:3, v/v) at room temperature. Experimental Protocols

General A reaction vessel equipped with a fritted disc, a solvent inlet, and a device to transfer in and out liquids under low nitrogen pressure was used to carry out solid-phase syntheses. This manual apparatus enables suspensions of resin to be stirred either by gently rocking the vessel on a shaker or by bubbling nitrogen through the fritted plate. Chemicals Aminomethylpolystyrene resin (0.56 mmol/g, NovaBiochem, La¨ ufelfingen, Switzerland) TentaGel S-NH2 resin (0.28 mmol/g, RappPolymere, Tu¨ bingen, Germany) Fmoc-Rink amide linker (NovaBiochem, La¨ ufelfingen, Switzerland) AmAbz(Fmoc) (16) (prepared according to Pascal et al.25 or commercially available from Senn Chemical AG, Dielsdorf, Switzerland) Use of AmAbz Building Blocks in the Solid-Phase Synthesis of Peptidomimetics: The Typical Example of Heptapeptide 19 25 Elongation. Aminomethylpolystyrene resin (1 g, 0.56 mmol) is first washed with DMF, CH2Cl2, TFA/CH2Cl2 (1:1, v/v), CH2Cl2, DIEA/ CH2Cl2 (1:20, v/v), CH2Cl2, and DMF four times each. Then, Fmoc-Rink linker (0.45 g, 0.84 mmol) is added to the resin with DMF (3 ml), BOP (0.37 g, 0.84 mmol), and DIEA (0.22 ml, 1.26 mmol) and the mixture is shaken for 90 min. The resin is filtered and washed with DMF, and then DIEA (0.29 ml, 1.68 mmol) and acetic anhydride (0.53 ml, 5.6 mmol) are added to cap unreacted amino groups. Except for Fmoc-Phe, the incorporation of the next amino acids is carried out as follows: (1) the Fmoc group is removed with piperidine/DMF (1:4, v/v) (1  1 min þ 3  3 min) and the resin is filtered and washed with DMF (4  1 min); (2) the N-protected amino acid building block (1.85 mmol) and HOBtH2O (0.26 g, 1.68 mmol) mmol) are then added to the resin using a minimum volume of DMF, then DIEA (0.44 ml, 2.52 mmol) and BOP (0.743 g, 1.68 mmol) are added and

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the mixture is shaken for 60 min. The resin is filtered and washed with DMF (4  1 min) and subjected to the next deprotection step or, at the end of the synthesis, it is filtered, washed with DMF, CH2Cl2, MeOH, 100% EtOH, and Et2O, and then dried. Acylation of the Arylamino Group. The resin is washed with DMF (4  1 min) and CH2Cl2 (4  1 min). A solution of Fmoc-Phe (0.716 g, 1.85 mmol) in CH2Cl2 (10 ml) is added to the resin then DIC (0.263 ml, 1.68 mmol) is added to the mixture. CH2Cl2 (10 ml) is added 5 min later because of the precipitation of a Fmoc-Phe-activated species and the reaction mixture is shaken for 3 h. Fmoc-Phe coupling is then repeated using a similar procedure except that DMF (6 ml) is added after 5 min and CH2Cl2 is evaporated off by nitrogen bubbling during the reaction. Release and Recovery of Peptide 19. The dried resin (0.21 g) is suspended in TFA/H2O/TIS (95:2.5:2.5, v/v/v, 8.4 ml) and the mixture is shaken for 3 h. The suspension is filtered and the resin is washed with TFA (2  2 ml). The filtrate is concentrated, diluted with Et2O (100 ml), and then extracted with water (2  20 ml). The combined aqueous layers are concentrated and freeze-dried to give peptide 19 as a white solid (20 mg). Preparation and Use of Dpr(Phoc) Linker Preparation of Boc-Dpr.30,36 A mixture of diacetoxyiodobenzene (24.16 g, 75 mmol), acetonitrile (100 ml), and water (100 ml) is stirred until almost complete dissolution. Acetic acid (8.6 ml, 150 mmol) is then added and solid Boc-Asn (11.61 g, 50 mmol) is introduced into the flask with acetonitrile (25 ml) and water (25 ml). The mixture is stirred at room temperature for 24 h. The phenyl iodide by-product is removed by extraction with t-BuOMe (2  100 ml). The aqueous layer is concentrated under reduced pressure and the solid residue is suspended in cold EtOH (100 ml), collected by filtration, and washed with cold EtOH then with Et2O, and dried under vacuum to give crude Boc-Dpr as a white crystalline solid (7.62 g, 75%). Preparation of Boc-Dpr(Phoc).30 Crude Boc-Dpr (4.52 g, 22.1 mmol) is dissolved in water (110 ml) with sodium bicarbonate (4.65 g, 55.3 mmol). The mixture is stirred vigorously at room temperature, while phenyl chloroformate (3.35 ml, 26.7 mmol) is added in five portions over 30 min. Stirring is continued for 4 h then the mixture is transferred into a separating 36

L.-h. Zhang, G. S. Kauffman, J. A. Pesti, and J. Yin, J. Org. Chem. 62, 6918 (1997); 63, 10085 (1998).

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funnel and washed with t-BuOMe (2  100 ml). The t-BuOMe layer is separated and the aqueous layer is acidified with 1 M NaHSO4 (33 ml) and extracted with ethyl acetate (2  75 ml). The combined extracts are washed with water and brine, then dried (Na2SO4) and concentrated under reduced pressure. The oily residue is diluted with CH2Cl2 (5 ml), then the solvent is evaporated under reduced pressure to give Boc-Dpr(Phoc) as a white solid foam (5.53 g, 77%). Preparation of Boc-Dpr(Phoc)–OSu. A solution of Boc-Dpr(Phoc) (11.06 g, 34.1 mmol) and HOSu (4.12 g, 35.8 mmol) in DME (20 ml) is
cooled to 0 then a solution of DCC in DME (15 ml) is added. The mixture

is stirred at 0 for 60 min and allowed to stand overnight at 4 . The precipitate is filtered off, washed with cold DME, and the filtrate is concentrated under reduced pressure. Toluene (100 ml) is added to the residue and crystallization is initiated by ultrasonic irradiation (15 s) and continued over
night at 4 . The solid is collected by filtration and washed with pentane. Recrystallization from toluene gives Boc-Dpr(Phoc)–OSu as a white solid (11.2 g, 78%). Preparation of the Resin Carrying Boc-Dpr(Phoc). The Tentagel resin (1 g, 0.28 mmol) is swollen in DMF (10 ml) for 30 min and then filtered and washed with DMF four times. A mixture of Boc-Sar (0.175 g, 0.93 mmol) HOBtH2O (0.129 g, 0.84 mmol) and DIC (0.132 ml, 0.84 mmol) is stirred for 5 min in CH2Cl2 (1 ml) then added to the resin with a minimum volume of DMF to allow stirring. DIEA (0.146 ml, 0.84 mmol) is added and the suspension is shaken for 60 min. The resin is filtered and washed with DMF and CH2Cl2 four times each and Boc-protecting groups are removed with TFA/CH2Cl2 (1:1, v/v) (5 ml, 1  1 min þ 1  30 min). Then the resin is filtered and washed with CH2Cl2 (4  1 min), neutralized with DIEA/ CH2Cl2 (1:20, v/v) (5 ml, 3  2 min), and washed with CH2Cl2 and DMF four times each. Boc-Dpr(Phoc)–OSu, 0.177 g (0.42 mmol) and HOBtH2O (0.064 g, 0.42 mmol) are added with DMF (2 ml). The suspension is shaken, then DIEA (0.073 ml, 0.42 mmol) is added and shaking is continued for 24 h at room temperature, then the resin is filtered and washed with DMF four times. Acetic anhydride (0.26 ml, 2.8 mmol) and pyridine/ CH2Cl2 (1:19, v/v) (5 ml) are added and allowed to react for 15 min to cap unreacted amino groups. The resin is filtered and washed with DMF, CH2Cl2, MeOH, EtOH, and Et2O four times each and dried under vacuum. Boc-protecting groups are removed with TFA/CH2Cl2 and the C-terminal residue of the target peptide is coupled to the resin (as an NBoc- or N-Fmoc-protected building block) using the Boc protocol described below. Then, elongation can be continued using either the Boc or the Fmoc protocols as follows.

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linkers and their applications

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Boc Protocol Deprotection and Coupling. The resin (0.28 mmol) is treated with TFA/CH2Cl2 (1:1, v/v) (5 ml, 1  1 min þ 1  30 min), then filtered and washed (1 min) with CH2Cl2, MeOH, and DMF four times each. The amino acid building block (0.84 mmol) is activated with HOBtH2O (0.129 g, 0.84 mmol) and DIC (0.132 ml, 0.84 mmol) by stirring for 5 min in CH2Cl2 (1 ml). Then the mixture is added to the resin with a minimum volume of DMF to allow stirring. DIEA (0.122 ml, 0.70 mmol) is added and the suspension is shaken for 30 min. Further DIEA is added (0.073 ml, 0.42 mmol) and shaking is continued for 30 min. The resin is filtered and washed with DMF then CH2Cl2 four times each. Fmoc Protocol Cyclization of the Linker. After the N-Fmoc-protected C-terminal residue has been coupled, the resin (0.28 mmol) is filtered, washed with DMF, and then repeatedly treated with a solution of PhONa-PhOH in DMF (7 ml, 10  20 min) prepared as follows: a mixture of phenol (0.264 g, 2.80 mmol) and 1 N NaOH (1.4 ml, 1.4 mmol) is concentrated under reduced pressure without heating; DMF (10 ml) is added to the residue then the solvent is evaporated under reduced pressure; phenol (0.033 g, 0.35 mmol) is added to the residue and the final volume of the solution is adjusted to 70 ml with DMF. The resin is filtered and washed with DMF four times. Deprotection and Coupling. The resin (0.28 mmol) is treated with piperidine/DMF (1:4, v/v) (5 ml, 1  1 min þ 3  3 min), then washed with DMF (4  1 min). A mixture of the N-Fmoc amino acid building block (0.84 mmol), HOBtH2O (0.129 g, 0.84 mmol), and DIC (0.132 ml, 0.84 mmol) is stirred for 5 min in CH2Cl2 (1 ml) then added to the resin with a minimum volume of DMF to allow stirring. DIEA (0.073 ml, 0.42 mmol) is added and the suspension is shaken for 30 min. Further DIEA is added (0.073 ml, 0.42 mmol) and shaking is continued for 30 min. The resin is filtered and washed with DMF four times.