Domino Process Achieves Site-Selective Peptide Modification with

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Domino Process Achieves Site-Selective Peptide Modification with High Optical Purity. Applications to Chain Diversification and Peptide Ligation Ivan Romero-Estudillo, and Alicia Boto J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00932 • Publication Date (Web): 07 Jul 2015 Downloaded from http://pubs.acs.org on July 8, 2015

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The Journal of Organic Chemistry

Domino Process Achieves Site-Selective Peptide Modification with High Optical Purity. Applications to Chain Diversification and Peptide Ligation. Ivan Romero-Estudillo, and Alicia Boto* Instituto de Productos Naturales y Agrobiología del CSIC, Avda. Astrofísico Francisco Sánchez 3, 38206-La Laguna, Tenerife, Spain [email protected] RECEIVED DATE Corresponding Author Footnote. Telephone: +34-922-260112 (Ext 267); Fax: +34 922260135.

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ACS Paragon Plus Environment


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Table of Contents Graphic

Abstract. The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes, but still is a difficult synthetic challenge. Herein we introduce natural hydroxyproline as convertible unit for the production of a variety of optically pure aminoacids, including expensive N-alkyl aminoacids, homoserine lactones and Agl lactams, and to achieve the mild, efficient and site-selective modification of peptides. A domino process is used to cleave the customizable Hyp unit under mild, metal-free conditions. Both terminal and internal positions can be modified, and similar customizable units can be differentiated. The resulting products possess two reactive chains which can be manipulated independently. The versatility and scope of this process is highlighted by its application to the ligation of two peptide chains, and the generation of peptides with several chains and peptides with conformational restrictions.

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Introduction The modification of peptides is a hot research topic,1 in order to provide new peptide drugs,1,2 probes for molecular imaging1,3 and peptide catalysts for green chemistry.4 The usual procedure to create libraries of potential peptide drugs or catalysts involves the synthesis de novo of each library member.1-4 To introduce each new amino acid, two steps are needed (deprotection of the terminal residue and a coupling step), and therefore, this serial process is costly in time and materials, particularly with large peptides. In addition, some peptides are quite difficult to obtain (eg. difficult sequences, macrocycles, etc), and producing each library member will require an extra effort. Finally, since for many activity studies only a small part of the sequence (active center, binding site) needs modification, a different approach to create peptide libraries would be desirable. In this alternative strategy, a certain position/s in a parent peptide is transformed, while the others remain unchanged (site-selective modification).5-7 In the tag-and-modify approach, a functional group in a residue (tag) is replaced or transformed into other ones. This strategy has allowed the modification of both peptides and proteins.8 A variation involves the use of customizable units, where the core of the starting aminoacid is transformed into a quite different one (eg. by cleavage of the lateral chain and replacement by new ones). 9-15 The simplest customizable unit is glycine, and thus, Kazmaier9 and Skrydstrup10 have reported their selective enolization and reaction with electrophiles. Other customizable units are aziridine carboxylic acids, and Van der Donk and Garner have described their opening by different nucleophiles.11 A problem in the site-selective modification of peptides is differentiating similar tags or customizable units. Thus, when a peptide has several glycine units, the selective enolization of just one residue is a real challenge. Important progress was achieved with the introduction of customizable units whose reactive groups could be protected orthogonally. For instance, Steglich and Skrydstrup reported the selective chain scission of serine or threonine residues in peptides.12 The residues with unprotected hydroxyl groups underwent lateral chain scission, while protected residues were not affected. Since 3



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many orthogonal protecting groups are available, different functionalizations at different times can be achieved. Recently, our group developed a sequential method for the oxidative radical scission of serine residues in peptides, followed by the addition of C- or P-nucleophiles (conversion 1→3, Scheme 1).13,14 Although good product yields were obtained, the stereoselectivity of the reaction ranged from excellent to low.13 In order to prepare peptides with consistently high optical purity, in a preliminary communication15 we introduced another customizable unit, natural and low-cost 4R-hydroxy-L-proline (Hyp). SCHEME 1. Site-selective conversion of customizable units Ser (previous work, left) and Hyp (this work, right). OH

O

HO 4 3 5

Z-HN

N H

R

X

COX

N Z-(aa)n

O

1

4 Z = carbamoyl, acyl, amino acyl, peptidyl X = O-alkyl, amino acyl, peptidyl R = H, alkyl, aryl PhI(OAc)2 I2, hv,

Pb(OAc)4 Scission-oxidation process

O Z-HN R

O + N H

X

COX

N+ Z-(aa)n

2 Nu

5 Addition of nucleophiles

AcO

Orthogonal "handles" AcO R

CHO

Nu

O Z-HN

O

N H

COX

Z-(aa)n O 6

3 P- and C-chains Unnatural residues Low or moderate d.e.

X

N

Unnatural units High optical purity

4


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When introduced in peptides 4 (Scheme 1), the Hyp unit could be selectively transformed using a mild, metal-free domino process. The scission reaction proceeded regioselectively through the C4-C5 bond,16 to yield the acyliminium intermediate 5,17 which retained the stereogenic -center. The addition of acetate ions from the reaction medium followed, yielding scission products 6 with two reactive handles, which could be manipulated independently. In this Paper, a significant extension of these studies will be reported, where different scission products 6 will be generated and transformed into a variety of optically pure compounds, from N-alkyl amino acids to unusual -alkyl and alkenyl glycines, and peptides with rigid Agl lactams that allow conformational constraints in the backbone. In addition, the versatility of this strategy will be highlighted by its application to the ligation of peptides, generating larger linear or branched derivatives. Results and Discussion Studies on the optical purity of the products during the scission and subsequent reductive amination steps. As commented before, the fragmentation of Hyp units through the C4-C5 bond should not affect the -stereogenic center, and the process would afford L-amino acids unless epimerization of the -center took place under the reaction conditions. In order to check this point, substrates where the Hyp unit was attached to a chiral moiety were prepared. When hydroxyproline (-)-menthyl carbamate 815 (Scheme 2) was treated with (diacetoxy)iodobenzene (DIB, PhI(OAc)2) and iodine, under irradiation with visible light at 26 oC, the 4-oxo-L-homoalanine derivative 10 was formed in 80% yield. To our satisfaction, a

single diastereomer was generated, supporting the hypothesis that under the mild

reaction conditions no epimerization occurred. Interestingly, no C3-C4 scission products were isolated, and also an N-acetoxymethyl group was formed, by reaction of the acyliminium intermediate with acetate ions from DIB. The scission reaction was repeated with the dipeptide Cbz-Phe-Hyp-OMe (9). In this case, the temperature was increased to favour substrate solubility, and to reduce reaction times, despite the 5



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heating could promote the epimerization of both residues. However, after 1 h, the reaction afforded the dipeptide 11 as a single diastereomer in 82% yield. SCHEME 2. Studies on the stereochemical integrity of the scission and reductive amination products. H-Hyp-OMe (7) ClC(O)O-menthyl or Cbz-Phe-OH, DIPEA, HBTU 87-91%

ClCOOMe, NaHCO3 (aq) THF, 94%

HO

MeO(O)C-Hyp-OMe (12) O

DIB, I2, h DCE, 80 oC, 1 h

N OMe Z

O 8 Z = COO-(-)menthyl 9 Z = (N-Cbz)-Phe DIB, I2, h , CH2Cl2, 2 h

AcO

MeO

OMe O

13 (81%)

O

AcO

O N

NH2-R NaBH4, MeOH

O N

OMe

Z

N R

10 Z = O

(80%)

HN MeO

O

11 Z =

(82%) O HN

O

O

14 R =

(77%)

15 R = CH2-Ph

(77%)

16 R =

(66%)

17 R =

(68%)

O O

Negligible epimerization; high optical purity

6


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The N-acetoxymethyl 4-oxo-L-homoalanine derivatives resulting from the scission could be valuable precursors of other compounds, by controlled manipulation of the reactive N- or -chains.18 A requisite is that such transformations proceed without epimerization of the stereogenic centers. Therefore, the reductive amination of the scission products with primary amines was studied. This reaction can provide high-profit -amino--lactams (Agl derivatives), as shown by the conversion of aldehyde 13 (Scheme 2) into lactams 1417. Two enantiomeric 1-(naphthyl)ethyl amines were used to check that no epimerization took place during the reductive amination-lactamization process, which afforded the diastereomeric compounds 16 (2S,1’R) and 17 (2S,1’S).15 After purification of both lactams, different diastereomer mixtures were prepared [(1’S):(1’R) 85:15, 90:10, 95:5, 98:2] and their NMR spectra was recorded. The detection limit was about dr 98:2. With these spectra, it was checked that the crude reductive amination mixture corresponded to (1’S):(1’R) ≥ 96:4 (see NMR series, Supporting Information) Creation of Agl units by reductive amination of the scission products, and application to peptide ligation. The reductive amination-lactamization process was also studied with aminoacids and peptides (Scheme 3). Thus, the aldehyde 13 reacted with H-Thr(tBu)-OMe to give lactam 18 in 59% yield, in spite of the steric hindrance of the amine. When peptide 19 was used, the tripeptide 20 was formed in 82% yield as a single diastereomer. The formation of -amino--lactams (Agl, Freidinger-Veber lactam) has proven very useful to rigidify peptide backbones, inducing conformations that mimic -turns.1a,19 These turns are often found in bioactive peptides which interact with their biological targets. Moreover, Agl-containing peptide drugs often display increased stability and bioavailability, and less undesired interactions. Therefore, the scission-reductive amination process was adapted to achieve a double goal: the ligation of small peptides and the concomitant formation of an Agl unit to introduce conformational constraints.

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SCHEME 3. Preparation of peptides with Agl lactams. CHO AcO

O N

MeO

H-Thr(tBu)-OMe, Et 3N, MeOH 26 o C, 1 h; then NaBH 4, 45 o C, 20 h

OMe O

CO2 Me OtBu

N

O

HN

O MeO

13

H2 N

18 (59%) 19 , MeOH Et 3N, 26 oC, 1 h; O then NaBH 4, o Ile-OMe 45 C, 20 h

( )3

CO2 Me

NH-Boc 19 1. H-Ile-OMe, DCM DIPEA, HBTU 0 to 26 oC, 87% 2. H2 , Pd/C, 93%

O

NH

( )2

NH Boc

N O

O Cbz-HN

( )3 21

OH NH-Boc

O

HN

OMe 20 (82%)

The process was studied with the aldehyde 11 (Scheme 4) which reacted with glycine methyl ester under reductive conditions, affording the Agl-containing tripeptide 22 with high optical purity. When the aldehyde 13 was treated with peptide 19 under similar conditions, the ligation of the two peptides proceeded with lactamization, affording the desired tetrapeptide 23 in 78% yield; no epimerization was detected.

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SCHEME 4. Ligation of peptides, forming a rigid Agl unit. O NH

HN O

O

N

O

OMe O

22

H-Gly-OMe, MeOH, Et3N, 26°C, 1h; then NaBH4, 45°C, 20 h, 64% AcO

O N

HN O

O

CO2Me

O 11

Boc-Lys-Ile-OMe (19) MeOH, Et3N, 26°C, 1h; then NaBH4, 45°C, 20 h, 78% OMe O

O

O

O

O

NH Boc

O NH

HN

NH

N

23

Increasing diversity by manipulation of the N- and -chain reactive handles. Another valuable unit in peptide chemistry is L-homoserine lactone, which reaches high market value as component of compounds affecting quorum sensing in bacteria, and thus, microbial virulence.20 This lactone could be readily obtained by reduction of 4-oxo-L-homoalanine derivatives and intramolecular cyclization (Scheme 5). Depending on the reaction conditions, different products can be obtained. For instance, the reduction of aldehyde 10 with triethylsilane under Lewis acid catalysis proceeded to give the desired lactone, but 9



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in addition, the N-acetoxymethyl moiety was reduced to a N-methyl group, affording compound 24 as a single diastereomer. The N-alkylated amino acids are valuable, since their introduction into peptides alters the hydrogen bonding pattern, favouring extended conformations. This allows modulation of the peptide activity and potency. The modified peptides also present increased bioavailability and stability to proteolysis.1a In spite of their usefulness, there are relatively few commercially available N-methyl aminoacids, and are quite expensive. This method allows the conversion of low-cost substrates into valuable N-substituted amino acids. SCHEME 5. Formation of valuable L-homoserine lactones, including N-methyl derivatives. CHO

AcO N O

O

O

Et3SiH

OMe

BF3OEt2

O N O

70%

10

O

O

24

O O

AcO

N

MeO

O

O

NaBH4

OMe

HN

MeOH

MeO

77%

13 OH N N Cbz O

CO2Me

O O 25

1. DIB, I2, h CH2Cl2, 26 oC 2. NaBH4, MeOH 51% (two steps)

H N N Cbz O

O O

27

26 O

AcO N N Cbz O

H CO2Me

28

By varying the reduction conditions, the N-acetoxymethyl group can be removed. Thus, when substrate 13 was treated with sodium borohydride in methanol, the homoserine lactone 25 was isolated 10


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in 77% yield. Similar reductive conditions were applied for the transformation of dipeptide 26 into the homoserine lactone derivative 27.21 Interestingly, the intermediate aldehyde 28 was not purified, but treated after aqueous work-up with sodium borohydride to give the final product. The selective manipulation of the -chain, without affecting the N-substituent, is also possible. In the example shown in Scheme 6 (transformation 29→31),15 the homoalanine side chain was extended using a Horner-Wadsworth-Emmons (HWE) reaction, which generated an expensive dehydrohomoglutamic acid derivative. To our satisfaction, both the fragmentation and the HWE reaction proceeded without epimerization to give compound 31 as a single diastereomer. In general, the orthogonal manipulation of the - and N-chains could remarkably increase product diversity, as will be shown in the next examples. SCHEME 6. Selective manipulation of the - chain by HWE reaction. OH

Fmoc

Fmoc HN

OAc

NH N

N OMe

O

O

O

29

CO2Me OMe

O 31

DIB, I 2 , h, CH 2Cl2

63%

NaH, THF

82%

(MeO) 2(O)P

CO2 Me

O O

OAc

NH N O

O

CHO OMe

30

Scission of Hyp units in internal positions, and ligation to amino acids or peptides. The scission of hydroxyproline units in internal positions represented an additional challenge, since peptide folding could increase the probability that the reactive O-radical derived from Hyp was drawn close to aromatic rings, carbonyl groups, or hydrogens on benzylic, tertiary or -carbons. In that case, side-reactions such as addition of the O-radical to multiple bonds or hydrogen abstraction from nearby positions could take place. However, if the scission was favored over side-reactions, a diversity of peptide libraries could be 11



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obtained. For instance, the conversion of customizable Hyp units into 4-amino homoalanines could be applied to the generation of antibiotic peptides,1,2 where a balance of non polar and cationic residues is crucial for antimicrobial activity while avoiding hemolysis.22-24 The cleavage of Hyp in internal positions and its conversion to 4-amino homoalanines was studied with a model tetrapeptide, which was prepared from compounds 7 and 3225 as shown in Scheme 7. SCHEME 7. Scission of Hyp units in internal positions for the preparation of antimicrobials. H-Hyp-OCH3 (7)

Boc-Pro-Ile-OMe (32)

1) Cbz-Phe-OH, DCM HBTU, DIPEA 2) NaOH, MeOH

TFA, DCM

OH H Cbz

N

HN O

N NH

O OH

O

O

33 (86%)

OCH3

34 (99%) DIPEA, HBTU, DCM HO N

N O O

HN

O

NH

O

O

OCH3

O 35 (89%) DIB, I2, h DCE, 80 oC 45 min, 64%

X AcO

N

N

HN

O O

O

O

NH O

OCH3

O 36 X = CHO O 37 X =

N

morpholine, Et3N, DCE; NaBH(OAc)3 86%

12


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The coupling of dipeptides 33 and 3426 afforded substrate 3515 in very good yield. The scission of the tetrapeptide 35 under treatment with DIB and iodine was promoted by heating, in part for solubility reasons. The reaction afforded the aldehyde 36 (64%), which was readily purified and converted into a 4-morpholino homoalanine derivative 37 using a reductive amination reaction (86%). The morpholinocontaining residue is used in commercial antimicrobials such as cobicistat.23 In order to study the selectivity of the scission when two or more Hyp units were present, a model peptide was prepared as shown in Scheme 8 (conversions 7→3927 and 39→40). SCHEME 8. Site-selective modification of one Hyp unit in an internal position.

In substrate 40 the internal Hyp residue had a free hydroxyl group, while the C-terminal Hyp unit was protected. When the scission reaction was carried out, only the unprotected Hyp residue was cleaved, affording the aldehyde 41 in high optical purity. In this case, no heating was necessary to promote the scission. The cleavage of the internal Hyp unit is interesting from a conformational standpoint, since it

13



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decreases the system rigidity, can alter elements of the secondary structure (such as turns) and finally, the presence or removal of the N-acetoxymethyl groups can influence the number of hydrogen bonds. On the other side, the attachment of the scission products to amino acids or peptides would allow the creation of new interactions or folding patterns.28 The ligation studies were performed with aldehyde 41 (Scheme 9), which underwent a reductive amination with proline benzyl ester, yielding the tetrapeptide 42 in 83% yield. SCHEME 9. Peptide ligation after selective scission of one Hyp unit in an internal position. H-Pro-OBn DCE, Et3 N, 26°C 10 min; then

CO2 Bn

N NaBH(OAc) 3 , 6 h AcO 83% Cbz

O

OTBS N

N

H N

O

O

OTBS

CO2 Me

42

AcO

Cbz

H N

N

N O

O

CO2 Me O Boc-HN

41

N H

CO2 Me

( )2 Boc-Lys-Ile-OMe MeOH, Et 3 N 26°C, 1h, then NaBH4 45°C, 18 h 75%

N

Z OTBS

Cbz

H N

N

HN O

ClCO2 Me, THF NaHCO3 (ac), 97%

O

CO2 Me

43 Z = H 44 Z = CO2Me

The reductive amination also allows ligation of two peptide chains. Thus, when the aldehyde 41 was treated with Boc-Lys-Ile-OMe under reductive conditions, the pentapeptide 43 was obtained in 76% yield. In order to reduce its polarity and facilitate its manipulation, the peptide was carbamoylated, affording compound 44 in 97% yield. The creation of libraries of peptides with several chains would 14


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facilitate the identification of drugs with multiple interactions with the biological target and/or decreased proteolysis in vivo, as well as the development of peptide catalysts with improved substrate recognition The applications of this methodology to create libraries of antimicrobial and catalytic peptides will be reported in due course. Conclusions. Natural and inexpensive hydroxyproline has been introduced as customizable unit for the generation of a diversity of amino acids with high optical purity, including valuable N-alkyl amino acids, homoserine lactones, Freidinger-Veber lactams, 4-aminohomoalanines, and -alkylglycines. Hydroxyproline underwent a mild, metal-free domino scission-oxidation-addition of acetate ions process, generating a residue with two reactive chains (the -lateral chain and the N-acetoxymethyl group) which can be transformed independently. The customizable unit was also used for the mild, site-selective modification of peptides, an strategy that allowed the creation of peptide libraries from a single (or a few) parent peptides, saving valuable time and materials in discovery processes. The reactive group in the Hyp unit can be protected with orthogonal groups, allowing differenciation between several customizable units in the same peptide. The free residues would be transformed, while protected ones would remain unchanged. The process is versatile, and thus, when the scission of C-terminal positions was followed by a reductive amination, a double goal was achieved: the ligation of small peptides and the concomitant formation of an Agl unit (Freidinger-Veber lactam) to introduce conformational constraints. When the scission was performed on internal positions of the peptide, the reductive amination provided potential antimicrobial compounds; moreover, the ligation of two peptide chains gave compounds capable of multiple interactions with biological or catalysis targets. On the other side, the manipulation of the N-acetoxymethyl moiety allowed the release of the free amino group group or the formation of N-methylated units, which in turn allows modulation of the peptide conformation and activity.

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Experimental Section.

General Remarks: General Methods. Commercially available reagents and solvents were analytical grade or were purified by standard procedures prior to use. All reactions involving air- or moisturesensitive materials were carried out under nitrogen atmosphere. Three alternative spray reagents for TLC analysis were used: (a) 0.5% vanillin in H2SO4-EtOH (4:1); (b) 0.25% ninhydrin in ethanol and (c) Fleet’s reagent [Ce(SO4)2 (0.5 g) and ammonium phosphomolybdate hydrate (2.5 g) in H2SO4 (5 mL) and water (65 mL)]. Once sprayed, the TLC was heated until development of color. Merck silica gel 60 PF254 and 60 (0.063-0.2 mm) were used for rotatory chromatography and column chromatography, respectively. Melting points were determined with a hot-stage apparatus and are uncorrected. Optical rotations were measured at the sodium line at ambient temperature (26 oC). Mass spectra were carried out using electrospray ionization techniques (ESI) or electronic impact (EI), the latter was determined at 70 eV using an ion trap mass analyzer. Nuclear Magnetic Resonance spectra were determined at 500 or 400 MHz for 1H NMR and 125.7 or 100 MHz for 13C NMR in the presence of tetramethylsilane (TMS) as internal standard, at 25 oC or 70 oC, as stated for each case. Due to rotamer equilibrium, the resolution of some NMR spectra at 26 oC was low (formation of broad bands); for those cases, the spectra at 70 oC are provided. 1H NMR references: CDCl3 (H 7.26), CD3OD (H 3.31); 13C NMR references: CDCl3 (C 77.0), CD3OD (C 49.0). Abbreviations (in the NMR spectra): br b, broad band; br d, broad doublet, etc The aminoacid and peptide derivatives H-Pro-OBn∙HCl, H-Trp-OEt∙HCl, H-Thr(tBu)-OMe∙HCl, HHyp-OMe∙HCl, H-Pro-OMe∙HCl, Boc-Val-OH, Cbz-Leu-OH, Cbz-Phe-OH and Boc-Thr(Bn)-Ala-PheOH are commercial reagents. N-[(1R,2S,5R)-Menthyloxycarbonyl]-4R-hydroxy-L-proline methyl ester (8):15 4R-hydroxy-Lproline methyl ester hydrochloride (0.600 g, 3.3 mmol) was added to a saturated aqueous NaHCO3 solution (10.0 mL). After cooling at 0 oC, a solution of ()-menthyl chloroformate (0.85 mL, 3.96 mmol) in THF (10.0 mL) was added. The mixture was allowed to reach 26 oC and stirred for 3 h. Then 16


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it was poured into water and extracted with EtOAc, and the organic layer was dried over sodium sulfate and filtered. The solvent was removed under vacuum and the residue was purified by column chromatography (hexane-EtOAc 80:20) to give menthyl carbamate (8) (1.014 g, 94%) as an oil: []D = 99.27 (c 0.37, CHCl3); 1H NMR (500 MHz, CDCl3, 70 oC): H 0.81 (d, J = 7.0 Hz, 3H), 0.800.91 (m, 2H), 0.89 (d, J = 5.9 Hz, 3H), 0.90 (3H, d, J = 6.9 Hz), 1.07 (m, 1H), 1.33 (m, 1H), 1.46 (m, 1H), 1.641.73 (m, 2H), 1.85 (br b, 1H, OH), 1.93 (m, 1H), 2.04 (m, 1H), 2.11 (ddd, J = 5.1, 7.0, 13.3 Hz, 1H), 2.27 (1H, m), 3.52 (m, 1H), 3.67 (dd, J = 4.7, 11.7 Hz, 1H), 3.71 (s, 3H), 4.44 (m, 1H), 4.48 (m, 1H), 4.55 (ddd, J = 4.4, 10.7, 11.0 Hz, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 16.9, 20.7, 21.9, 24.2, 26.8, 31.5, 34.5, 38.8, 41.7, 47.7, 52.0, 54.8, 57.9, 69.4, 75.9, 154.2, 173.1. IR (CHCl3): 3417, 1746, 1694, 1420 cm–1. HRMS (EI) calcd for C17H29NO5 (M+) 327.2046, found 327.2053. Anal. Calcd for C17H29NO5: C, 62.36; H, 8.93; N, 4.28; found: C, 62.30; H, 8.80; N, 4.35. N-[N-(Benzyloxycarbonyl)phenylalanyl]-4R-hydroxy-L-proline methyl ester (9): A solution of Cbz-Phe-OH (0.700 g, 2.34 mmol) and H-Hyp-OMe•HCl (0.552 g, 3.04 mmol) in dichloromethane (30 mL) at 0°C, was treated with DIPEA (0.8 mL, 4.6 mmol) and HBTU (0.978 g, 2.57 mmol). After 1 h, the reaction mixture was allowed to reach room temperature and stirred for 1 h. Then it was poured into saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with 5% aqueous HCl and with water, dried over anhydrous sodium sulfate, filtered and evaporated under vacuum. The residue was purified by column chromatography on silica gel (hexane/EtOAc, 40:60), yielding the dipeptide (9) (0.868 g, 87%), as a white foam: []D = 38.0 (c 0.43, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 1.98 (ddd, J = 4.7, 7.9, 12.9 Hz, 1H), 2.08 (br b, 1H), 2.23 (br dd., J = 9.5, 10.7 Hz, 1H), 2.95 (dd, J = 6.3, 13.6 Hz, 1H), 3.10 (dd, J = 7.6, 13.9 Hz, 1H), 3.22 (dd, J = 4.1, 11.0 Hz, 1H), 3.69 (br b, 1H), 3.73 (s, 3H), 4.40 (m, 1H), 4.63 (dd, J = 7.9, 8.2 Hz, 1H), 4.67 (m, 1H), 5.01 (d, J = 12.3 Hz, 1H), 5.08 (d, J = 12.3 Hz, 1H), 5.53 (br b, 1H), 7.207.35 (m, 10H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 37.7, 39.1, 52.1, 54.0, 55.2, 57.9, 67.1, 70.3, 127.0, 128.0, 128.5, 129.7, 136.2, 136.5, 170.8, 172.1; one (C) signal corresponding to the carbamate group was not clearly observed. IR 17



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(CHCl3) 3405, 1742, 1722, 1710, 1641, 1631 cm–1. HRMS (EI) calcd for C23H26N2O6 (M+) 426.1791, found 426.1800. General procedure for the scission of 4-hydroxyproline derivatives: A solution of the substrate (1 mmol) in dichloromethane (10 mL) was treated with (diacetoxyiodo)benzene (DIB, 0.644 g, 2.0 mmol) and iodine (0.127 g, 0.5 mmol). The reaction mixture was irradiated with visible light (80 W tungstenfilament lamp) at 26 oC until disappearance of the starting material (2.5-4 h); then was poured into 10% aqueous Na2S2O3, and extracted with CH2Cl2. The organic layer was dried over sodium sulfate and filtered, then the solvent was removed under vacuum and the residue was purified by column chromatography (hexane/EtOAc), yielding N-acetoxymethyl-4-oxohomoalanine derivatives. (2S) N-(Acetoxymethyl)-N-[(1R,2S,5R)-menthyloxycarbonyl]-4-oxo-L-homoalanine methyl ester (10): Obtained from hydroxyproline derivative (8) (0.327 g, 1.0 mmol) according to the General Scission Procedure. After 2.5 h, the reaction mixture was extracted and purified by column chromatography (hexane/EtOAc 85:15), yielding compound (10) (0.308 g, 80%) as a yellowish oil: []D = 95.7 (c 0.87, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.78 (d, J = 6.9 Hz, 3H), 0.86 (m, 1H), 0.89 (d, J = 7.3 Hz, 3H), 0.91 (d, J = 6.7 Hz, 3H), 0.981.10 (m, 2H), 1.37 (m, 1H), 1.47 (m, 1H), 1.641.72 (m, 2H), 1.83 (m, 1H), 2.02 (s, 3H), 2.03 (m, 1H), 2.95 (m, 1H), 3.31 (dd, J = 6.0, 17.9 Hz, 1H), 3.70 (s, 3H), 4.63 (br ddd, J = 4.1, 10.7, 10.8 Hz, 1H), 4.86 (dd, J = 6.6, 6.7 Hz, 1H), 5.40 (d, J = 11.1 Hz, 1H), 5.43 (brd, J = 11.4 Hz, 1H), 9.75 (s, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 16.3, 20.5, 20.6, 21.8, 23.7, 26.4, 31.4, 34.2, 41.1, 44.8, 47.4, 52.4, 55.5, 72.4, 77.2, 154.8, 170.2, 170.4, 197.9. IR (CHCl3): 1741, 1707, 1458, 1420 cm–1. HRMS (EI) calcd for C18H28NO7 [M+  Me] 370.1866, found 370.1861. Anal. Calcd for C19H31NO7: C, 59.20; H, 8.11; N, 3.63; found: C, 59.21; H, 8.16; N, 3.70. N-Acetoxymethyl-N-(N-benzyloxycarbonyl-L-phenylalanyl)-4-oxo-L-homoalanine methyl ester (11): A solution of the dipeptide Cbz-Phe-Hyp-OMe (9) (86.0 mg, 0.2 mmol) in dry dichloroethane (4 mL) was treated with DIB (130.0 mg, 0.4 mmol) and iodine (51.0 mg, 0.2 mmol). The mixture was stirred at 80 oC for 1 h, under irradiation with visible light. Then it was poured into 10% aqueous 18


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Na2S2O3 and extracted with CH2Cl2. After drying and filtering the organic layer in the usual way, the solvent was evaporated under vacuum, and the residue was purified by rotatory chromatography (hexane/EtOAc 70:30), affording the aldehyde 11 (80.0 mg, 82%) as a white foam: []D = 28.6 (c 0.42, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 1.97 (s, 3H), 2.78 (dd, J = 7.2, 18.5 Hz, 1H), 2.95 (dd, J = 6.3, 13.5 Hz, 1H), 3.03 (dd, J = 7.7, 13.6 Hz, 1H), 3.27 (dd, J = 5.2, 18.4 Hz, 1H), 3.62 (s, 3H), 4.76 (dd, J = 6.0, 6.2 Hz, 1H), 5.02 (dd, J = 7.9, 7.8 Hz, 1H), 5.06 (d, J = 12.4 Hz, 1H), 5.09 (d, J = 12.4 Hz, 1H), 5.20 (br d, J = 12.1 Hz, 1H), 5.305.45 (m, 2H), 7.107.40 (m, 10H), 9.62 (s, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 20.5, 39.9, 44.0, 52.5, 52.6, 55.7, 67.0, 72.0, 127.2, 128.0, 128.1, 128.5, 128.6, 129.5, 135.9, 136.6, 155.5, 169.6, 170.2, 173.3, 197.9. IR (CHCl3) 3327, 1744, 1721, 1670, 1522 cm–1. HRMS (EI) calcd for C23H25N2O6 (M+  CO2Me) 425.1713, found 425.1698; calcd for C7H7 ([PhCH2]+) 91.0548, found 91.0550. Anal. Calcd for C25H28N2O8: C, 61.98; H, 5.83; N, 5.78; found: C, 61.63; H, 5.99; N, 5.94. N-[(Methyloxycarbonyl]-4R-hydroxy-L-proline methyl ester (12) has been previously reported.29 (2S)-N-Methoxycarbonyl-N-(acetoxymethyl)-4-oxo-L-homoalanine methyl ester (13): Obtained from (4R) N-methoxycarbonyl-4-hydroxy-L-proline methyl ester (12) (0.203 g, 1.0 mmol) according to the General Scission Procedure. After 2.5 h, the reaction mixture was extracted and purified by column chromatography (hexane/EtOAc 80:20), yielding compound (13) (0.212 g, 81%), as a yellowish oil: []D = 84.5 (c 0.35, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 2.04 (s, 3H), 3.02 (m, 1H), 3.30 (dd, J = 5.7, 18.3 Hz, 1H), 3.72 (s, 3H), 3.75 (s, 3H), 4.90 (dd, J = 5.7, 5.7 Hz, 1H), 5.43 (s, 2H), 9.75 (s, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 20.7, 44.7, 52.7, 53.4, 55.6, 72.5, 155.6, 170.2, 170.6, 198.0. IR (CHCl3): 1728, 1480, 1441 cm–1. HRMS (EI) calcd for C8H12NO6 [M+  CH2CHO] 218.0665, found 218.0657. Anal. Calcd for C10H15NO7: C, 45.98; H, 5.79; N, 5.36; found: C, 45.67; H, 5.91; N, 5.46. General procedure for the reductive amination-lactamization: A solution of aldehyde (0.20 mmol) in dry methanol (3.0 mL) was treated with the amine (0.28 mmol) and triethylamine (38.0 µL, 19



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0.27 mmol). After 1 h at 26 ºC, sodium borohydride (10.0 mg, 0.26 mmol) was added and the reaction mixture was warmed to 45 oC for 20 h. Then the mixture was allowed to reach room temperature and poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated under vacuum and the residue was purified by rotatory chromatography (hexane/EtOAc), yielding the corresponding -amino lactam. (S) 1-Allyl-3-(N-methoxycarbonyl)amino-2-pyrrolidinone (14): A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with allylamine (20.0 µL, 0.27 mmol) following the General procedure of reductive amination-lactamization. The residue was purified by rotatory chromatography (hexane/EtOAc 40:60), yielding lactam 14 (31.0 mg, 77%) as a white solid: m.p. 9294 ºC (EtOAc/hexane); []D = 9.2 (c 0.3, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 1.89 (m, 1H), 2.61 (m, 1H), 3.253.35 (m, 2H), 3.67 (s, 3H), 3.86 (dd, J = 6.2, 15.2 Hz, 1H), 3.93 (dd, J = 6.0, 15.2 Hz, 1H), 4.22 (dd, J = 9.2, 9.3 Hz, 1H), 5.18 (dddd, J = 1.3, 1.3, 1.4, 8.8 Hz, 1H), 5.205.22 (m, 1H), 5.52 (br b, 1H), 5.70 (m, 1H).

13

C NMR (125.7 MHz, CDCl3, 26 oC): C 27.7, 43.6, 45.8, 52.2,

52.8, 118.4, 131.6, 157.0, 171.8. IR (CHCl3) 3426, 1722, 1695, 1513 cm–1. HRMS (EI) calcd for C9H14N2O3 [M+] 198.1004, found 198.1011; calcd for C7H9NO (M+ NH2CO2Me) 123.0684, found 123.0682. Anal. Calcd for C9H14N2O3: C, 54.53; H, 7.12; N, 14.13; found: C, 54.35; H, 7.20; N, 14.36. (S) 1-Benzyl-3-(N-methoxycarbonyl)amino-2-pyrrolidinone (15): A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3 mL) was treated with benzylamine (30.0 µL, 0.28 mmol) following the General procedure of reductive amination-lactamization. The residue was purified by rotatory chromatography (hexane/EtOAc 50:50), yielding lactam 15 (39.0 mg, 77%) as a white solid: m.p. 121123 ºC (EtOAc/hexane); []D = 7.96 (c 0.33, CHCl3). 1H NMR (500 MHz, CDCl3 with TMS, 26 oC): H 1.86 (m, 1H), 2.61 (m, 1H), 3.183.25 (m, 2H), 3.70 (s, 3H), 4.26 (m, 1H), 4.46 (d, J = 15 Hz, 1H), 4.51 (d, J = 14.7 Hz, 1H), 5.45 (br b, 1H, NH), 7.23 (br.d., J = 7.8 Hz, 2H), 7.277.36 (m, 3H).

13

C

NMR (125.7 MHz, CDCl3, 26 oC): C 27.7, 43.3, 47.1, 52.2, 52.8, 127.7, 128.0, 128.7, 135.7, 157.0, 171.9. IR (CHCl3) 1722, 1697, 1510 cm–1. HRMS (EI) calcd for C13H16N2O3 [M+] 248.1161, found 20


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248.1162; calcd for C11H11NO (M+  NH2CO2Me) 173.0841, found 173.0846. Anal. Calcd for C13H16N2O3: C, 62.89; H, 6.50; N, 11.28; found: C, 63.11; H, 6.75; N, 10.95. (1'R,3S) 1-[1'-(2-Naphthalenyl)ethyl)-3-(N-methoxycarbonyl)amino-2-pyrrolidinone (16): A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with (R)-(+)-1-(2naphthyl)ethylamine (48.0 mg, 0.28 mmol) and triethylamine (38.0 µL, 0.27 mmol). After stirring for 1 h at 26 ºC, sodium borohydride (10.0 mg, 0.26 mmol) was added, and then the mixture was heated to 45 ºC for 20 h. After cooling to 26 oC, it was poured into water and extracted with EtOAc. The organic layer was dried and filtered as usual, the solvent was evaporated under vacuum and the residue was purified by rotatory chromatography (hexane/EtOAc 50:50), yielding lactam 16 (42.0 mg, 66%) as a white solid: m.p. 148150 ºC (EtOAc/hexane); []D = +217.9 (c 0.36, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 1.66 (d, J = 7.1 Hz, 3H), 1.85 (m, 1H), 2.52 (m, 1H), 2.85 (ddd, J = 6.8, 6.8, 9.8 Hz, 1H), 3.32 (dd, J = 9.3, 9.7 Hz, 1H), 3.69 (s, 3H), 4.22 (m, 1H), 5.56 (br.b., 1H), 5.64 (ddd, J = 7.0, 7.0, 7.1 Hz, 1H), 7.35 (dd, J = 1.5, 8.6 Hz, 1H), 7.457.52 (m, 2H), 7.72 (s, 1H), 7.787.88 (m, 3H).

13

C

NMR (125.7 MHz, CDCl3, 26 oC): C 15.9, 27.7, 39.4, 50.0, 52.2, 53.2, 125.5, 126.1, 126.3, 127.6, 127.9, 128.5, 132.8, 133.1, 136.9, 157.0, 171.6. IR (CHCl3): 3426, 3018, 1721, 1688, 1511 cm–1. HRMS (EI) calcd for C18H20N2O3 [M+] 312.1474, found 312.1472. Anal. Calcd for C18H20N2O3: C, 69.21; H, 6.45; N, 8.97; found: C, 68.96; H, 6.42; N, 8.79. (1'S,3S)

1-[1'-(2-Naphthalenyl)ethyl)-3-(N-methoxycarbonyl)amino-2-pyrrolidinone

(17):

Obtained from aldehyde 13 (53.0 mg, 0.20 mmol) and (S)-()-1-(2-naphthyl)ethylamine (48.0 mg, 0.28 mmol) according to the previous procedure. After work-up and rotatory chromatography purification (hexane/EtOAc 50:50), lactam 17 was obtained (45.0 mg, 68%) as a slighty yellowish solid: m.p. 194197 ºC (EtOAc/hexane); []D = 207.8 (c 0.34, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 1.63 (d, J = 7.1 Hz, 3H), 1.70 (m, 1H), 2.58 (m, 1H), 2.99 (dd, J = 9.3, 9.4 Hz, 1H), 3.28 (ddd, J = 6.4, 10.0, 10.0 Hz, 1H), 3.70 (s, 3H), 4.30 (m, 1H), 5.50 (br. b., 1H), 5.62 (ddd, J = 7.0, 7.1, 7.1 Hz, 1H), 7.39 (dd, J = 1.7, 8.6 Hz, 1H), 7.457.51 (m, 2H), 7.73 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.797.84 (m, 2H). 13C 21



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NMR (125.7 MHz, CDCl3, 26 oC): C 16.2, 28.2, 39.3, 49.8, 52.3, 53.3, 125.4, 125.5, 126.2, 126.4, 127.6, 128.0, 128.6, 132.8, 133.2, 137.1, 157.1, 171.6. IR (CHCl3): 3424, 1724, 1692 cm–1. HRMS (EI) calcd for C18H20N2O3 [M+] 312.1474, found 312.1474. Experiments to determine the stereoselectivity of the process. The pure isomers 16 and 17 do not only have different spectroscopic and physical data, but also present different Rf (0.45 for 16 and 0.39 for 17. Therefore, in case that both isomers were formed in significant amounts, they could be separated by chromatography. Careful purification of the mixture yielded only one diastereomer in each case. However, in the crude product mixture, traces of a minor diastereomer could be detected (dr ≥ 96:4). In order to check the accuracy of the 1H NMR to determine the diastereomer ratio by integration of the signals, mixtures of the purified isomers 16 and 17 were prepared, by weighting different amounts of the pure isomers, and mixing them in CDCl3. 1H NMR spectra of the 85:15, 90:10, 95:5 and 98:2 (16):(17)-mixtures were recorded, and we checked that the integral ratio corresponded to the real isomer ratio. In the 98:2 mixture the minor isomer could hardly be detected by 1H NMR, due to NMR sensitivity limit (although the sensitivity is often superior to other detection techniques).30 In the next section (NMR spectra) records of these experiments are displayed. (2S,3S)-3-(terc-Butoxy)-2-((S)-3-((methoxycarbonyl)amino)-2-oxo-1-pyrrolidinyl)butanoate (18): A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with HThr(OtBu)-OMe•HCl (64.0 mg, 0.28 mmol), following the General procedure of reductive aminationlactamization. The residue was purified by rotatory chromatography (hexane/EtOAc 50:50), yielding lactam 18 (39.0 mg, 59%) as a yellowish oil. []D = +34.0 (c 0.32, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 1.12 (d, J = 6.3 Hz, 3H), 1.15 (s, 9H), 1.88 (m, 1H), 2.64 (ddd, J = 6.0, 6.7, 12.3 Hz, 1H), 3.52 (ddd, J = 6.1, 10.6, 10.7 Hz, 1H), 3.69 (s, 3H), 3.70 (s, 3H), 3.86 (br dd., J = 8.8, 10.7 Hz, 1H), 4.31 (m, 1H), 4.42 (dddd, J = 2.9, 6.3, 6.5, 6.6 Hz, 1H), 4.77 (d, J = 2.9 Hz, 1H), 5.16 (br b, 1H). 13

C NMR (125.7 MHz, CDCl3, 26 oC): C 20.7, 28.7, 29.8, 44.4, 51.8, 52.2, 52.6, 60.4, 68.3, 74.2,

157.1, 169.7, 173.7. IR (CHCl3) 1729, 1703, 1698 cm–1. HRMS (EI) calcd for C14H23N2O6 [M+  Me]

22


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315.1556, found 315.1551; calcd for C8H10N2O4 (M+  OMe  CH(Me)OtBu) 198.0641, found 198.0640. Anal. Calcd for C15H26N2O6: C, 54.53; H, 7.93; N, 8.48; found: C, 54.60; H, 8.08; N, 8.42. Synthesis

of

compound

(19)

and

its

precursor

(45).

N2-(tert-Butoxycarbonyl)-N6-

(benzyloxycarbonyl)-L-lysyl-L-isoleucine methyl ester (45): A solution of Boc-Lys(Cbz)-OH (21) (1.00 g, 2.6 mmol) and H-Ile-OMe·HCl (0.614 g, 3.38 mmol) in dichloromethane (30 mL) at 0 °C was treated with DIPEA (0.89 mL, 5.1 mmol), and HBTU (1.096 g, 2.89 mmol). The reaction mixture was stirred at 0 °C for 1 h and then was allowed to reach room temperature and stirred for 1 h more. Then it was poured over saturated aqueous NaHCO3, and the organic layer was extracted with EtOAc and washed with 10% aqueous HCl. The organic layer was dried and filtered as usual, and the solvent was removed under vacuum. The residue was purified by column chromatography on silica gel (hexane/EtOAc, 60:40), yielding dipeptide 45 (1.145 g, 87%), as a white foam: []D = 8.0 (c 0.36, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 0.89 (dd, J = 6.5, 6.7 Hz, 3H), 0.91 (d, J = 6.4 Hz, 3H), 1.18 (m, 1H), 1.341.47 (m, 3H), 1.42 (s, 9H), 1.471.57 (m, 2H), 1.62 (m, 1H), 1.82 (m, 1H), 1.89 (m, 1H), 3.123.22 (m, 2H), 3.68 (s, 3H), 4.10 (m, 1H), 4.55 (d, J = 5.0, 8.6 Hz, 1H), 5.08 (br b, 1H, NH), 5.09 (br s, 2H), 5.23 (br b, 1H, NH), 6.69 (br b, 1H, NH), 7.257.35 (m, 5H). 13C NMR (125.7 MHz, CDCl3, 26 oC): C 11.5, 15.5, 22.5, 25.0, 28.3, 29.4, 31.6, 37.7, 40.5, 52.1, 54.3, 56.5, 66.6, 80.0, 128.1, 128.5, 136.7, 155.8, 156.6, 172.0, 172.3. IR (CHCl3) 3324, 1722, 1703, 1694, 1666, 1530 cm–1. HRMS (EI) calcd for C26H41N3O7 [M+] 507.2945, found 507.2939; calcd for C7H7 ([PhCH2]+) 91.0548, found 91.0545. Anal. Calcd for C26H41N3O7: C, 61.52; H, 8.14; N, 8.28; found: C, 61.15; H, 8.42; N, 8.14. N2-(tert-Butoxycarbonyl)-L-lysyl-L-isoleucine methyl ester (19): A solution of the dipeptide 45 (1.00 g, 1.97 mmol) in methanol (30 mL) was treated with 10% Pd/C (200 mg), and the reaction mixture was stirred under hydrogen for 15 h. Then it was filtered through celite, and the solvent was evaporated under vacuum, affording the dipeptide 19 (0.685 g, 93%), as a yellowish oil: []D = 15.0 (c 0.35, CHCl3). 1H NMR (500 MHz, CD3OD, 26 oC): H 0.93 (d, J = 7.2 Hz, 3H), 0.95 (dd, J = 6.4, 6.8 Hz, 3H), 1.27 (m, 1H), 1.401.55 (m, 5H), 1.46 (s, 9H), 1.61 (m, 1H), 1.75 (m, 1H), 1.91 (m, 1H), 2.68 23



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(dd, J = 7.1, 7.1 Hz, 2H), 3.73 (s, 3H), 4.09 (br dd, J = 6.5, 7.1 Hz, 1H), 4.41 (d, J = 5.9 Hz, 1H). 13C NMR (125.7 MHz, CD3OD, 26 oC): C 11.6, 16.0, 24.1, 26.3, 28.7, 32.8, 33.0, 38.5, 42.1, 52.4, 55.8, 58.1, 80.6, 157.9, 173.5, 175.3. IR (CHCl3) 3327, 1734, 1699, 1684, 1654 cm–1. HRMS (EI) calcd for C18H35N3O5Na ([M  Na]+) 396.2474, found 396.2463. N2-(Methoxycarbonyl)-2-(4-(2-oxo-1-pyrrolidinyl)butyl)-L-glycyl-L-isoleucine methyl ester (20): A solution of aldehyde 13 (53.0 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with Boc-LysIle-OMe (19) (105.0 mg, 0.28 mmol), following the General procedure of reductive aminationlactamization. The residue was purified by rotatory chromatography (hexane/EtOAc, 10:90), affording lactam 20 (84.0 mg, 82 %) as a yellowish oil: []D = 12.5 (c 0.38, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.86 (dd, J = 7.4, 7.5 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H), 1.16 (m, 1H), 1.32 (m, 2H), 1.39 (m, 9H), 1.40 (m, 1H), 1.54 (m, 2H), 1.58 (m, 1H), 1.751.87 (m, 3H), 2.54 (m, 1H), 3.223.33 (m, 4H), 3.62 (s, 3H), 3.67 (s, 3H), 4.00 (m, 1H), 4.16 (br dd, J = 8.2, 8.3 Hz, 1H), 4.47 (dd, J = 5.1, 8.5 Hz, 1H), 5.13 (br d, J = 7.6 Hz, 1H), 5.52 (br b, 1H), 6.68 (br d, J = 8.2 Hz, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 11.3, 15.4, 22.4, 25.1, 26.4, 27.8, 28.2, 31.4, 37.7, 42.4, 43.8, 51.7, 52.0, 52.9, 54.6, 56.6, 79.9, 155.7, 157.0, 171.8, 172.0. IR (CHCl3) 3306, 2965, 1721, 1708, 1690, 1681, 1668, 1530 cm– 1

. HRMS (EI) calcd for C24H42N4O8 (M+) 514.3003, found 514.3013. Methyl 2-(3S-[N-(benzyloxycarbonyl)phenylalanyl]amino-2-oxo-1-pyrrolidinyl)acetate (22): A

solution of aldehyde 11 (50.0 mg, 0.10 mmol) in dry methanol (2.0 mL) was treated with H-GlyOMe•HCl (18.0 mg, 0.14 mmol) and Et3N (20.0 L, 14 mmol), following the General Procedure for reductive amination-lactamization. After the usual work-up, the residue was purified by rotatory chromatography (hexane/EtOAc, 30:70), affording product 22 (30.0 mg, 64%), as a yellowish foam: []D = 6.3 (c 0.37, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 1.84 (m, 1H), 2.64 (ddd, J = 6.1, 6.4, 12.5 Hz, 1H), 3.06 (dd, J = 7.0, 14.1 Hz, 1H), 3.11 (dd, J = 6.4, 14.0 Hz, 1H), 3.34 (dd, J = 8.4, 9.2 Hz, 1H), 3.46 (ddd, J = 6.8, 9.3, 9.4 Hz, 1H), 3.72 (s, 3H), 3.95 (d, J = 17.5 Hz, 1H), 4.12 (d, J = 17.5 Hz, 1H), 4.31 (m, 1H), 4.44 (ddd, J = 6.9, 6.9, 7.5 Hz, 1H), 5.05 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.4 24


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Hz, 1H), 5.30 (d, J = 7.1 Hz, 1H), 6.41 (br b, 1H), 7.177.32 (m, 10H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 27.6, 38.7, 44.5, 44.9, 51.4, 52.2, 56.3, 67.1, 127.1, 128.0, 128.1, 128.5, 128.7, 129.5, 136.4, 136.5, 155.9, 168.6, 171.6, 172.3. IR (CHCl3) 3296, 1744, 1703, 1698, 1680, 1537 cm–1. HRMS (EI) calcd for C24H27N3O6 (M+) 453.1900, found 453.1885; calcd for C7H7 ([PhCH2]+) 91.0548, found 91.0545. Anal. Calcd for C24H27N3O6: C, 63.56; H, 6.00; N, 9.27; found: C, 63.47; H, 6.28; N, 9.56. 2-(4-(3R-(N-Benzyloxycarbonyl-L-phenylalanyl)amino-2-oxo-1-pyrrolidinyl)butyl)-2-N-(tertbutoxycarbonyl)-L-glycyl-L-isoleucine methyl ester (23): A solution of the aldehyde 11 (50.0 mg, 0.10 mmol) in dry methanol (2.0 mL), was treated with Boc-Lys-IIe-OMe (19) (53.0 mg, 0.14 mmol) and Et3N (20.0 L, 0.14 mmol), following the General Procedure for reductive amination-lactamization. After the usual work-up, the residue was purified by rotatory chromatography (hexane/EtOAc, 80:20), affording product 23 (59.0 mg, 78%), as a yellowish foam: []D = 18.0 (c 0.36, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.88 (dd, J = 7.4, 7.6 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H), 1.20 (m, 1H), 1.301.50 (m, 3H), 1.42 (s, 9H), 1.51-1.69 (m, 3H), 1.78-1.90 (m, 3H), 2.54 (m, 1H), 3.03 (dd, J = 7.2, 14 Hz, 1H), 3.14 (dd, J = 6.0, 14 Hz, 1H), 3.18 (m, 1H), 3.21-3.31 (m, 2H), 3.42 (m, 1H), 3.70 (s, 3H), 4.04 (m, 1H), 4.30 (m, 1H), 4.49 (m, 1H), 4.52 (dd, J = 5.1, 8.5 Hz, 1H), 5.01 (d, J = 12.5 Hz, 1H), 5.08 (d, J = 12.4 Hz, 1H), 5.18 (br b, 1H, NH), 5.52 (br b, 1H, NH), 6.73 (br d, J = 7.7 Hz, 1H), 6.91 (br b, 1H, NH), 7.16-7.33 (m, 10H);

13

C NMR (125.7 MHz, CDCl3, 70 oC): C 11.4, 15.5, 22.3, 25.2, 26.3,

27.1, 28.3, 31.4, 37.8, 38.8, 42.2, 44.0, 51.7, 51.9, 54.8, 56.7, 66.9, 80.1, 126.8, 127.8, 127.9, 128.4, 128.5, 129.4, 136.5, 136.6, 155.8, 171.6, 171.9, 172.0, 172.0. IR (CHCl3) 3306, 1712, 1694, 1682, 1668, 1652, 1660, 1538 cm–1. HRMS (EI) calcd for C39H55N5O9Na (M+  Na) 760.3879, found 760.3885. Anal. Calcd for C39H55N5O9: C, 63.48; H, 7.51; N, 9.49; found: C, 63.54; H, 7.71; N, 9.80. N-[(1R,2S,5R)-menthyloxycarbonyl]-N-methyl-L-homoserine lactone (24): To a solution of aldehyde 10 (39.0 mg, 0.10 mmol) in dry dichloromethane (2.0 mL) were added BF3·OEt2 (25.0 µL, 0.2 mmol) and Et3SiH (40.0 µL, 0.25 mmol). The mixture was stirred at 26 ºC for 16 h, and then was poured into saturated aqueous NaHCO3 and extracted with dichloromethane. After drying and evaporating, the residue was purified by rotatory chromatography (hexane/EtOAc, 80:20), yielding the 25



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N-methyl homoserine lactone 24 (21.0 mg, 70%) as a yellowish oil: []D = 60.3 (c 0.34, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.81 (d, J = 7.0 Hz, 3H), 0.87 (m, 1H), 0.91 (d, J = 6.7 Hz, 3H), 0.92 (d, J = 7.0 Hz, 3H), 0.98 (ddd, J = 11.4, 11.7, 12 Hz, 1H), 1.08 (dddd, J = 3.2, 12.6, 12.9, 13.0 Hz, 1H), 1.39 (br dd, J = 11.0, 12.0 Hz, 1H), 1.50 (m, 1H), 1.661.73 (m, 2H), 1.92 (m, 1H), 2.09 (m, 1H), 2.38 (m, 1H), 2.45 (m, 1H), 2.92 (s, 3H), 4.23 (ddd, J = 7.0, 9.5, 9.8 Hz, 1H), 4.43 (ddd, J = 2.6, 9.2, 9.2 Hz, 1H), 4.63 (ddd, J = 4.4, 10.7, 11.1 Hz, 1H), 4.71 (br dd, J = 9.2, 9.8 Hz, 1H).

13

C NMR (125.7

MHz, CDCl3, 70 oC): C 16.6, 20.8, 21.9, 24.0, 26.0, 26.6, 31.5, 32.4, 34.5, 41.6, 47.7, 56.4, 65.1, 76.6, 156.1, 173.4. HRMS (EI) calcd for C16H27NO4 [M+] 297.1940, found 297.1946. Anal. Calcd for C16H27NO4: C, 64.62; H, 9.15; N, 4.71; found: C, 64.39; H, 9.14; N, 4.67. N-(Methoxycarbonyl)-L-homoserine lactone (25): A solution of aldehyde 13 (53 mg, 0.20 mmol) in dry methanol (3.0 mL) was treated with sodium borohydride (10.0 mg, 0.26 mmol) and stirred at 26 o

C for 4 h. Then the reaction mixture was poured into water and extracted with EtOAc. The organic

layer was dried, filtered and concentrated under vacuum. The residue was purified by rotatory chromatography (hexane/EtOAc, 60:40), affording lactone 25 (25.0 mg, 77%), as a white solid: 1H NMR (500 MHz, CDCl3, 26 oC): H 2.22 (m, 1H), 2.74 (m, 1H), 3.70 (s, 3H), 4.25 (m, 1H), 4.20-4.30 (m, 2H), 5.44 (br b, 1H, NH). 13C NMR (125.7 MHz, CDCl3, 26 oC): C 30.3, 50.5, 52.6, 65.7, 156.7, 175.0. HRMS (EI) calcd for C6H9NO4 (M+) 159.0532, found 159.0531; calcd for C5H9NO2 (M+  CO2) 115.0633, found 115.0634. Anal. Calcd for C6H9NO4: C, 45.28; H, 5.70; N, 8.80; found: C, 45.13; H, 5.80; N, 8.56. N-(N-Benzyloxycarbonyl-L-prolyl)-4R-hydroxy-L-proline methyl ester (26): A solution of CbzPro-OH (0.600 g, 2.41 mmol) and H-Hyp-OMe·HCl (0.479 g, 2.65 mmol) in dichloromethane (30.0 mL) at 0 °C, was treated with DIPEA (0.82 mL, 4.71 mmol), HOBt·H2O (0.404 g, 2.64 mmol) and EDAC (0.506 g, 2.64 mmol). After 1 h at 0 oC, the reaction mixture was allowed to reach room temperature and stirred for 20 h. Then it was poured into saturated aqueous NaHCO3 and extracted with EtOAc. The organic layer was washed with 10% aqueous HCl and with water, dried over anhydrous sodium sulfate, filtered and evaporated under vacuum. The residue was purified by column 26


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chromatography on silica gel (hexane/EtOAc, 40:60), yielding the dipeptide Cbz-Pro-Hyp-OMe (26) (0.753 g, 83%), as a white foam: []D = 98.9 (c 0.18, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC) 3:1 rotamer mixture: H 1.88 (m, 1H), 1.96 (ddd, J = 4.6, 8.5, 13.3 Hz, 1H), 1.982.20 (m, 3H), 2.34 (m, 1H), 3.50 (m, 1H), 3.543.68 (m, 2H), 3.70/3.71 (s, 3H), 4.01 (d, J = 10.9 Hz, 1H), 4.434.53 (m, 2H), 4.70 (dd, J = 8.3, 8.3 Hz, 1H), 5.03/5.04 ([d, J = 12.5 Hz/ d, J = 12.4 Hz], 1H), 5.10/5.14 ([d, J = 12.3 Hz/ d, J = 12.5 Hz], 1H), 7.30-7.34 (m, 5H).

13

C NMR (125.7 MHz, CDCl3, 26 oC): C 23.5/24.3,

29.3/30.1, 37.0/37.6, 46.9/47.2, 52.3, 54.4/54.9, 57.6, 57.9, 67.0/67.2, 70.3/70.5, 127.7, 128.0, 128.5, 136.5/136.9, 154.2/155.2, 171.3/171.6, 172.6/172.9. IR (CHCl3) 3440, 1744, 1684, 1658, 1449, 1436 cm–1. HRMS (EI) calcd for C19H24N2O6 (M+) 376.1634, found 376.1628; calcd for C7H7 ([OCH2Ph]+) 91.0548, found 91.0552. Anal. Calcd for C19H24N2O6: C, 60.63; H, 6.43; N, 7.44; found: C, 60.31; H, 6.53; N, 7.28. N-(N-Benzyloxycarbonyl-L-prolyl)-L-homoserine lactone (27): Obtained from the dipeptide (26) (0.376 g, 1.0 mmol) using the General procedure of radical scission-oxidation. After 4 h, the reaction mixture underwent the usual work-up, yielding a gum which contained the aldehyde intermediate [N(N-benzyloxycarbonyl-L-prolyl)-N-acetoxymethyl-4-oxo-L-homoalanine

methyl

ester (28)]. This

aldehyde was not purified, but dissolved in dry methanol (8 mL) and treated with sodium borohydride (50.0 mg, 1.2 mmol). The reaction mixture was warmed to 45 °C for 20 h, and then was cooled to room temperature, poured into water and extracted with EtOAc. The organic layer was dried, filtered and concentrated as usual, and the residue was purified by rotatory chromatography (hexane/EtOAc, 50:50), affording the lactone 27 (0.169 g, 51% for the two steps), as a yellowish foam: []D = 68.8 (c 0.2, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 1.89 (m, 1H), 1.942.14 (m, 3H), 2.28 (m, 1H), 2.65 (m, 1H), 3.483.58 (m, 2H), 4.20 (ddd, J = 6.3, 9.3, 10.6 Hz, 1H), 4.324.44 (m, 3H), 5.13 (d, J = 12.4 Hz, 1H), 5.18 (d, J = 12.4 Hz, 1H), 6.95 (br b, 1H, NH), 7.277.40 (m, 5H).

13

C NMR (125.7 MHz,

CDCl3, 70 oC): C 24.3, 29.8, 47.3, 49.2, 60.6, 65.7, 67.5, 128.0, 128.1, 128.5, 136.5, 172.5, 174.5; one signal, corresponding to the carbamate, was not clearly observed. IR (CHCl3) 3419, 1783, 1729, 1688 27



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cm–1. HRMS (EI) calcd for C17H20N2O5 (M+) 332.1372, found 332.1375; calcd for C7H7 ([CH2Ph]+) 91.0548, found 91.0545. N-(N-Fluorenylmethyloxycarbonyl-L-leucyl)-4R-hydroxy-L-proline methyl ester (29): To a solution of Fmoc-Leu-OH (700 mg, 1.98 mmol) and H-Hyp-OMe·HCl (400 mg, 2.20 mmol) in dichloromethane (30 mL) at 0°C were added DIPEA (0.67 mL, 3.85 mmol), HOBt (333 mg, 2.17 mmol) and EDAC (417 mg, 2.17 mmol). The reaction mixture was stirred at 0°C for 1 h and then was allowed to reach 26 oC and was stirred for other 20 h. Then it was poured into saturated aqueous NaHCO3, and the organic layer was washed with 10% aqueous HCl. After usual drying and solvent evaporation, the residue was purified by column chromatography (hexane/EtOAc, 60:40), yielding the dipeptide 29 (771 mg, 81%), as a white foam: []D = 58.13 (c 0.16, CHCl3). 1H NMR (400 MHz, CDCl3, 70 oC): H 0.96 (d, J = 6.7 Hz, 3H, Me), 0.98 (d, J = 6.5 Hz, 3H), 1.501.65 (m, 2H), 1.75 (m, 1H), 2.01 (m, 1H), 2.31 (br dd, J = 8.2, 13.4 Hz, 1H), 2.89 (br b, 1H, OH), 3.603.75 (m, 1H), 3.72 (s, 3H), 3.91 (d, J = 11.1 Hz, 1H), 4.17 (dd, J = 7.2, 7.5 Hz, 1H), 4.254.40 (m, 2H), 4.454.55 (m, 2H), 4.66 (dd, J = 8.2, 8.4 Hz, 1H), 5.59 (d, J = 8.5 Hz, 1H), 7.29 (dd, J = 7.4, 7.5 Hz, 2H), 7.38 (dd, J = 7.5, 7.5 Hz, 2H), 7.557.57 (m, 2H), 7.74 (d, J = 7.5 Hz, 2H).

13

C NMR (100.6 MHz, CDCl3, 26 oC): C

22.0, 23.1, 24.5, 37.5, 41.6, 47.1, 50.9, 52.2, 55.2, 57.6, 67.2, 70.3, 119.9, 125.1, 127.1, 127.7, 141.3, 143.7/143.8, 156.5, 171.8, 172.4. IR (CHCl3): 3430, 1745, 1711, 1650, 1511 cm–1. HRMS (EI) calcd for C27H32N2O6 [M+], 480.2260, found 480.2242. Anal. Calcd for C27H32N2O6: C, 67.48; H, 6.71; N, 5.83; found: C, 67.78; H, 6.67; N, 5.55. N-Acetoxymethyl-N-(N-fluorenylmethyloxycarbonyl-L-leucyl)-4-oxo-L-homoalanine

methyl

ester (30): Obtained from dipeptide 29 (0.480 g, 1.0 mmol) according to the General Scission Procedure. After 4 h, the reaction mixture was extracted and purified by column chromatography (hexane/EtOAc 70:30), yielding compound 30 (0.339 g, 63%), as a yellowish gum: []D = 35.74 (c 0.15, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 0.94 (d, J = 6.7 Hz, 3H), 1.00 (d, J = 6.6 Hz, 3H), 1.51 (m, 2H), 1.68 (m, 1H), 2.09 (s, 3H), 3.03 (dd, J = 7.5, 18.8 Hz, 1H), 3.39 (dd, J = 5.3, 18.6 Hz, 1H), 3.66 (s, 3H), 4.22 (dd, J = 7.1, 7.2 Hz, 1H), 4.36 (dd, J = 7.1, 10.2 Hz, 1H), 4.41 (dd, J = 7.2, 10.5 28


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Hz, 1H), 4.804.92 (m, 2H), 5.34 (d, J = 8.9 Hz, 1H), 5.46 (d, J = 12.2 Hz, 1H), 5.64 (d, J = 12.3 Hz, 1H), 7.31 (dd, J = 7.5, 7.5 Hz, 2H), 7.40 (dd, J = 7.3, 7.6 Hz, 2H), 7.59 (d, J = 7.5 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 9.75 (s, 1H). 13C NMR (125.7 MHz, CDCl3, 26 oC): C 20.6, 21.7, 23.3, 24.7, 42.7, 43.9, 47.2, 49.8, 52.7, 55.5, 67.0, 72.1, 120.0, 125.1, 127.1, 127.7, 141.3, 143.8, 155.9, 169.7, 170.4, 174.7, 198.4. IR (CHCl3): 3430, 1745, 1724, 1669, 1570 cm–1. HRMS (EI) calcd for C29H34N2O8 [M+] 538.2315, found 538.2332. Anal. Calcd for C29H34N2O8: C, 64.67; H, 6.36; N, 5.20; found: C, 64.51; H, 6.19; N, 5.54. Dimethyl

N-Acetoxymethyl-N-(N-fluorenylmethyloxycarbonyl-L-leucyl)-N-acetoxymethyl-4,5-

dehydro-L-homoglutamate (31): A solution of methyl 2-(dimethoxyphosphoryl)acetate (27.0 mg, 0.15 mmol) in dry THF (1.0 mL) was slowly added to a suspension of sodium hydride (60 % in mineral oil, 6.0 mg, 0.15 mmol) in dry THF (1.5 mL) at 10 °C. After stirring for 45 minutes, a solution of aldehyde 30 (54.0 mg, 0.10 mmol) in dry THF (1 mL) was added dropwise, and the mixture was stirred at 10 °C for 35 min. Then it was poured into water and extracted with diethyl ether. The organic layer was dried, filtered and evaporated as usual, and the residue was purified by rotatory chromatography (hexane/EtOAc, 80:20), yielding product 31 (49.0 mg, 82%) as a yellowish syrup: []D = 22.30 (c 0.38, CHCl3). 1H NMR (500 MHz, CDCl3, 26 oC): H 0.94 (d, J = 6.7 Hz, 3H), 0.98 (d, J = 6.6 Hz, 3H), 1.421.56 (m, 2H), 1.70 (m, 1H), 2.10 (s, 3H), 2.92 (dd, J = 7.3, 7.4 Hz, 2H), 3.67 (s, 3H), 3.68 (s, 3H), 4.21 (dd, J = 7.2, 7.2 Hz, 1H), 4.34 (dd, J = 7.0, 10.6 Hz, 1H), 4.40 (dd, J = 7.4, 10.6 Hz, 1H), 4.55 (dd, J = 7.0, 8.3 Hz, 1H), 4.83 (ddd, J = 4.2, 9.1, 9.4 Hz, 1H), 5.34 (d, J = 9.1 Hz, 1H), 5.43 (d, J = 12.4 Hz, 1H), 5.55 (d, J = 12.4 Hz, 1H), 5.85 (d, J = 15.6 Hz, 1H), 6.80 (ddd, J = 7.6, 8.1, 15.6 Hz, 1H), 7.30 (dd, J = 7.5, 7.5 Hz, 2H), 7.39 (dd, J = 7.4, 7.5 Hz, 2H), 7.58 (d, J = 7.5 Hz, 2H), 7.75 (d, J = 7.6 Hz, 2H). 13

C NMR (125.7 MHz, CDCl3, 26 oC): C 20.6, 21.6, 23.2, 24.7, 31.6, 42.9, 47.2, 49.9, 51.4, 52.6, 59.6,

67.1, 71.7, 120.0, 124.0, 125.1, 127.0, 127.7, 141.3, 143.8, 143.9, 156.5, 166.3, 169.9, 170.5, 174.9. IR (CHCl3): 3431, 1745, 1721, 1674, 1511 cm–1. HRMS (EI) calcd for C32H38N2O9 [M+] 594.2577, found 594.2573. Anal. Calcd for C32H38N2O9: C, 64.63; H, 6.44; N, 4.71; found: C, 64.75; H, 6.68; N, 4.88. 29



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N-(N-Benzyloxycarbonyl-L-phenylalanyl)-L-trans-4-hydroxyproline (33): A solution of Cbz-PheOH (0.60 g, 2.0 mmol) in CH2Cl2 (20 mL) at 0 oC was treated with H-Hyp-OMe∙HCl (0.473 g, 2.60 mmol), DIPEA (0.7 mL, 4.0 mmol) and HBTU (0.834 g, 2.20 mmol). The reaction mixture was stirred at 0°C for 1 h and then was allowed to reach 26 oC and stirred for 3 h, before being poured into saturated aqueous NaHCO3. The organic layer was washed 10% aqueous HCl and dried as usual. After solvent removal under vacuum, the residue was purified by column chromatography (hexane/EtOAc, 40:60), yielding dipeptide Cbz-Phe-Hyp-OMe (0.755 g), as a white foam [MS (EI): 426 (1) [M+]; HRMS: calcd. for C23H26N2O6 426.1791, found 426.1800]. The dipeptide was dissolved in MeOH (20 mL) and the solution was cooled to 0 oC, and treated with 2N aqueous NaOH (8 mL). The reaction mixture was was allowed to reach 26 oC and stirred for other 3 h, then was poured into 10% aqueous HCl and extracted with EtOAc. The organic layer was dried and evaporated as usual, yielding the dipeptide Cbz-Phe-Hyp-OH (33) (0.709 g, 86% for the two steps): []D = 38.8 (c 0.38, MeOH). 1H NMR (500 MHz, CD3OD, 70 oC): H 2.05 (ddd, J = 5.0, 7.6, 13.2 Hz, 1H), 2.17 (m, 1H), 2.86 (dd, J = 7.9, 14 Hz, 1H), 3.07 (dd, J = 6.1, 14 Hz, 1H), 3.50 (dd, J = 4.4, 10.7 Hz, 1H), 3.67 (m, 1H), 4.36 (m, 1H), 4.54 (dd, J = 7.9, 8.0 Hz, 1H), 4.65 (br dd, J = 6.7, 6.8 Hz, 1H), 4.98 (d, J = 12.6 Hz, 1H), 5.03 (d, J = 12.6 Hz, 1H), 7.117.16 (m, 10H). 13C NMR (125.7 MHz, CDCl3, 26 oC): C 38.5, 39.1, 55.6, 56.1, 59.5, 67.8, 70.9, 127.7, 128.7, 128.9, 129.4, 130.7, 138.2, 172.9, 175.0; a signal corresponding to the carbamate was not clearly observed. HRMS (EI) calcd for C22H24N2O6 [M+] 412.1634, found 412.1654. Anal. Calcd for C22H24N2O6: C, 64.07; H, 5.87; N, 6.79; found: C, 64.06; H, 6.24; N, 6.72. N-(N-Benzyloxycarbonyl-L-phenylalanyl-L-hydroxyprolyl-L-prolyl)-L-isoleucine methyl ester (35): The dipeptide Boc-Pro-IIe-OMe (32)25 (0.470 g, 1.4 mmol) was dissolved in CH2Cl2 (15.0 mL) at 0°C and treated with trifluoroacetic acid (15.0 mL). The reaction mixture was allowed to reach 26 oC and stirred for 2 h, then the volatiles were removed under vacuum. The crude dipeptide H-Pro-IIe-OMe (34)26 was not purified, but dissolved in CH2Cl2 (20.0 mL). The solution was cooled to 0 °C and treated with dipeptide Cbz-Phe-Hyp-OH (33) (0.435 g, 1.05 mmol), DIPEA (0.50 mL, 2.88 mmol) and HBTU (0.440 g, 1.16 mmol), and stirred for 1 h. The reaction mixture was allowed to reach 26 oC and stirred 30


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for other 3 h, then was poured into saturated aqueous NaHCO3, and afterwards the organic layer was washed with 10% aqueous HCl. After usual drying and solvent removal, the residue was purified by column chromatography (CH2Cl2/MeOH 95:5), yielding the tetrapeptide Cbz-Phe-Hyp-Pro-IIe-OMe (35) (0.596 g, 89%), as a white foam: []D = 74.3 (c 0.34, CHCl3). 1H NMR (500 MHz, CDCl3, 70 o

C): H 0.90 (d, J = 7.0 Hz, 3H), 0.92 (dd, J = 6.7, 7.3 Hz, 3H), 1.18 (m, 1H), 1.45 (m, 1H), 1.802.40

(m, 7H), 2.90 (dd, J = 6.6, 13.9 Hz, 1H), 3.12 (dd, J = 6.4, 14.1 Hz, 1H), 3.48 (br d, J = 14.2 Hz, 1H), 3.54 (m, 1H), 3.70 (m, 1H), 3.72 (s, 3H), 3.95 (m, 1H), 4.50 (m, 1H), 4.54 (m, 1H), 4.604.75 (m, 2H), 4.83 (br dd, J = 7.3, 7.3 Hz, 1H), 5.00 (d, J = 12.3 Hz, 1H), 5.08 (d, J = 12.3 Hz, 1H), 5.42 (br b, 1H), 7.14 (br b, 1H), 7.167.40 (m, 10H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 11.4, 15.5, 25.2, 27.2, 37.6, 37.9, 38.7, 47.6, 51.8, 53.9, 55.6, 56.8, 60.1, 67.0, 70.7, 126.9, 127.9, 128.1, 128.5, 128.7, 129.8, 136.1, 136.6, 156.0, 170.5, 171.0, 171.5, 172.1. HRMS (ESI) calcd for C34H44N4O8Na [M+ + Na] 659.3057, found 659.3062. Anal. Calcd for C34H44N4O8: C, 64.13; H, 6.97; N, 8.80; found: C, 64.44; H, 7.23; N, 8.87. N-(N-Benzyloxycarbonyl-L-phenylalanyl-[N-(acetoxymethyl)-4-oxo-L-homoalanyl]-L-prolyl)-Lisoleucine methyl ester (36). Method A: A solution of the tetrapeptide Cbz-Phe-Hyp-Pro-Ile-OMe (35) (64.0 mg, 0.1 mmol) in dry CH2Cl2 (5 mL) was treated with (diacetoxyiodo)benzene (64.0 mg, 0.2 mmol) and iodine (13.0 mg, 0.05 mmol). The reaction mixture was stirred at 26 oC for 4 h, under irradiation with visible light (80 W tungsten-filament lamp), then was extracted and evaporated as usual. The residue was purified by column chromatography (hexane/EtOAc, 50:50), affording the aldehyde derivative 36 (21.0 mg, 30%), as a foam. Method B: Similar to Method A, but the solvent was replaced by dry dichloroethane (5.0 mL) and the reaction mixture was stirred at 80 oC for 45 min under irradiation with visible light (80 W tungsten-filament lamp). After cooling to 26 oC, and the usual workup, the residue was purified as in Method A, affording the aldehyde 36 (44.0 mg, 64%) as a white foam: []D = 55.6 (c = 0.48, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.88 (d, J = 7.0 Hz, 3H), 0.91 (dd, J = 7.3, 7.6 Hz, 3H), 1.17 (m, 1H), 1.43 (m, 1H), 1.79 (m, 1H), 1.801.90 (m, 2H), 1.96 (s, 3H), 2.03 (m, 1H), 2.36 (m, 1H), 2.68 (br d, J = 18.0 Hz, 1H), 2.96 (dd, J = 6.4, 13.3 Hz, 1H), 3.08 (dd, J = 31



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7.9, 13.3 Hz, 1H), 3.23 (dd, J = 9.5, 17.7 Hz, 1H), 3.32 (m, 1H), 3.67 (m, 1H), 3.70 (s, 3H), 4.464.50 (m, 2H), 4.95 (ddd, J = 7.3, 7.6, 7.9 Hz, 1H), 5.05 (d, J = 12.3 Hz, 1H), 5.09 (d, J = 12.3 Hz, 1H), 5.26 (br b, 1H), 5.31 (d, J = 12.6 Hz, 1H), 5.58 (d, J = 12.0 Hz, 1H), 5.65 (m, 1H), 6.93 (br b, 1H), 7.17 (d, J = 6.3 Hz, 2H), 7.207.38 (m, 8H), 9.68 (br s, 1H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 11.3, 15.5, 20.6, 24.8, 25.3, 28.0, 37.7, 39.6, 44.4, 47.0, 51.7, 52.6, 56.9, 60.8, 67.2, 68.9, 127.3, 128.1, 128.2, 128.6, 128.7, 129.5, 129.6, 135.7, 136.4, 155.5, 169.3, 170.4, 172.0, 198.4; a carbon signal was not clearly observed. HRMS (ESI) calcd for C36H46N4O10Na [M+ + Na] 717.3105, found 717.3112. Anal. Calcd for C36H46N4O10: calcd. C, 62.23; H, 6.67; N, 8.06; found: C, 62.27; H, 6.85; N, 7.70. N-(N-Benzyloxycarbonyl-L-phenylalanyl-[N-(acetoxymethyl)-4-morpholino-L-homoalanyl]-Lprolyl)-L-isoleucine methyl ester (37). A solution of aldehyde 36 (0.10 g, 0.14 mmol) in dry dichloroethane (2.5 mL) was treated with morpholine (16.0 µL, 0.18 mmol) and triethylamine (25.0 µL, 0.18 mmol). After 10 min at 26 ºC, sodium (triacetoxy)borohydride (47.0 mg, 0.22 mmol) was added and the reaction mixture was stirred for 2.5 h. Then the mixture was extracted as usual, and the residue was purified by rotatory chromatography (hexane/EtOAc 10:90), yielding the tetrapeptide 37 (94.0 mg, 86%) as a yellowish syrup: []D = 36.8 (c = 0.13, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.87 (d, J = 7.0 Hz, 3H), 0.88 (dd, J = 7.3, 7.6 Hz, 3H), 1.18 (m, 1H), 1.43 (m, 1H), 1.771.93 (m, 4H), 1.95 (s, 3H), 1.962.10 (m, 2H), 2.26 (m, 1H), 2.272.65 (m, 6H), 2.90 (dd, J = 6.7, 13.6 Hz, 1H), 3.07 (dd, J = 7.0, 13.9 Hz, 1H), 3.34 (m, 1H), 3.66 (m, 1H), 3.68 (s, 3H), 3.673.75 (m, 4H), 4.41 (br d, J = 5.7 Hz, 1H), 4.47 (dd, J = 5.4, 8.2 Hz, 1H), 4.91 (ddd, J = 7.3, 7.3, 7.8 Hz, 1H), 5.01 (d, J = 12.3 Hz, 1H), 5.04 (d, J = 12.3 Hz, 1H), 5.30 (d, J = 8.2 Hz, 1H), 5.37 (br d, J = 11.8 Hz, 1H), 5.46 (m, 1H), 5.64 (d, J = 12.3 Hz, 1H), 7.05 (br b, 1H), 7.107.35 (m, 10H).

13

C NMR (125.7 MHz, CDCl3, 70 oC): C 11.3,

15.5, 20.6, 25.0, 25.3, 27.1, 28.0, 37.6, 39.6, 47.3, 51.7, 52.4, 52.6, 53.4, 54.3, 56.8, 60.5, 66.6, 67.0, 68.9, 127.1, 127.9, 128.1, 128.4, 128.5, 129.4, 135.8, 136.4, 155.4, 169.4, 170.7, 172.2, 173.6. IR (CHCl3): 3695, 3431, 1738, 1673, 1603, 1508 cm–1. HRMS (TOF) calcd for C40H56N5O10 [M+ + H] 766.4027, found 766.4033. Anal. Calcd for C40H55N5O10: calcd. C, 62.73; H, 7.24; N, 9.14; found: C, 62.64; H, 7.29; N, 9.20. 32


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N-Benzyloxycarbonyl-L-valyl-4R-hydroxy-L-proline methyl ester (38): A solution of H-HypOMe·HCl (0.940 g, 5.17 mmol) and Cbz-Val-OH (1.00 g, 3.97 mmol) and in dichloromethane (20.0 mL) at 0 °C was treated with DIPEA (1.3 mL, 7.47 mmol) and HBTU (1.657 g, 4.37 mmol). After 1 h, the reaction mixture was allowed to reach room temperature and stirred for 1 h. Then it was poured into saturated aqueous NaHCO3, and extracted with EtOAc. The organic layer was washed with 10% aqueous HCl, dried and concentrated as usual. The residue was purified by column chromatography on silica gel (hexane/EtOAc 30:70), yielding the dipeptide Cbz-Val-Hyp-OMe (38)27 (1.276 g, 85%), as a white foam: []D = 70.24 (c 0.41, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.94 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 1.96-2.06 (m, 2H), 2.27 (br dd, J = 10.3, 11.0 Hz, 1H), 2.79 (br b, 1H, OH), 3.67 (m, 1H), 3.69 (s, 3H), 3.86 (m, 1H), 4.25 (dd, J = 7.5, 7.8 Hz, 1H), 4.47 (m, 1H), 4.65 (dd, J = 8.2, 8.3 Hz, 1H), 5.02 (d, J = 12.4 Hz, 1H), 5.09 (d, J = 12.4 Hz, 1H), 5.53 (br b, 1H, NH), 7.257.31 (m, 5H). 13C NMR (125.7 MHz, CDCl3, 70 oC): C 17.6, 19.0, 31.2, 37.6, 51.9, 55.5, 57.7, 57.8, 67.0, 70.2, 127.8, 128.0, 128.4, 136.5, 156.7, 171.2, 172.3. IR (CHCl3) 3320, 1746, 1702, 1633, 1524 cm–1. HRMS (EI) calcd for C19H26N2O6 (M+), 378.1791, found 378.1796; calcd for C7H7 ([PhCH2]+) 91.0548, found 91.0546. Anal. Calcd for C19H26N2O6: C, 60.30; H, 6.93; N, 7.40; found: C, 60.36; H, 7.17; N, 7.65. N-Benzyloxycarbonyl-L-valyl-4R-hydroxy-L-proline (39): The dipeptide Cbz-Val-Hyp-OMe (38) (1.20 g, 3.17 mmol) was added to a 2N solution of KOH in 2:8 H2O:MeOH (5.0 mL) at 0 °C. The reaction mixture was allowed to reach room temperature, stirred at 26 oC for 2 h, then poured into 10% aqueous HCl and extracted with EtOAc. The organic layer was dried and concentrated in the usual way, affording the acid Cbz-Val-Hyp-OH (39)27 (1.096 g, 95%) as a white foam: []D = 66 (c 0.33, MeOH). 1

H NMR (500 MHz, CD3OD, 70 oC): H 0.97 (d, J = 6.8 Hz, 3H), 1.03 (d, J = 6.8 Hz, 3H), 2.052.14

(m, 2H), 2.28 (br dd, J = 9.0, 9.6 Hz, 1H), 3.78 (d, J = 4.3, 10.6 Hz, 1H), 3.82 (m, 1H), 4.28 (d, J = 7.2 Hz, 1H), 4.50 (m, 1H), 4.56 (dd, J = 8.1, 8.2 Hz, 1H), 5.11 (d, J = 12.7 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 7.287.38 (m, 5H). 13C NMR (125.7 MHz, CD3OD, 70 oC): C 18.3, 19.5, 32.2, 38.6, 56.6, 59.4, 59.5, 67.9, 71.0, 128.8, 129.0, 129.5, 138.2, 173.3, 175.1; a (C) signal, corresponding to the carbamate, 33



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was not clearly observed. HRMS (EI) calcd for C18H24N2O6, (M+) 364.1634, found 364.1640. Anal. Calcd for C18H24N2O6: C, 59.33; H, 6.64; N, 7.69; found: C, 59.09; H, 7.01; N, 7.69. N-Benzyloxycarbonyl-L-valyl-4R-hydroxy-L-prolyl-4R-(terc-butyldimethyl)silyloxy-L-proline methyl ester (40): A solution of Cbz-Val-Hyp-OH (39) (0.548 g, 1.50 mmol) and H-Hyp(TBS)-OMe31 (0.509 g, 1.95 mmol) in CH2Cl2 (20.0 mL) at 0°C was treated with DIPEA (0.4 mL, 2.28 mmol) and HBTU (0.827 g, 2.18 mmol). After 1 h, the reaction mixture was allowed to reach room temperature and stirred for 1 h. Then it was poured into saturated aqueous NaHCO3, and extracted with EtOAc. The organic layer was washed with 10% aqueous HCl, dried and concentrated as usual. The residue was purified by column chromatography on silica gel (hexane/EtOAc 30:70), yielding the tripeptide CbzVal-Hyp-Hyp(TBS)-OMe (40) (0.729 g, 81%), as a white foam: []D = 66 (c 0.38, MeOH). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.08 (s, 3H), 0.09 (s, 3H), 0.89 (s, 9H), 0.93 (d, J = 6.6 Hz, 3H), 1.00 (d, J =6.8 Hz, 3H), 2.022.19 (m, 4H), 2.24 (m, 1H), 2.46 (br b, 1H), 3.70 (s, 3H), 3.713.80 (m, 3H), 3.91 (m, 1H), 4.24 (dd, J = 7.6, 7.9 Hz, 1H), 4.514.56 (m, 2H), 4.63 (dd, J = 4.8, 8.8 Hz, 1H), 4.81 (dd, J = 7.9, 7.9 Hz, 1H), 5.04 (d, J = 12.4 Hz, 1H), 5.12 (d, J = 12.3 Hz, 1H), 5.45 (br b, 1H), 7.267.36 (m, 5H);

13

C NMR (125.7 MHz, CD3OD, 70 oC): C 4.9, 17.7, 17.9, 19.3, 25.7, 31.1, 37.1, 37.5, 52.0,

54.2, 55.8, 56.5, 57.5, 57.8, 67.0, 70.5, 70.6, 127.7, 127.9, 128.4, 136.5, 156.7, 170.7, 170.8, 172.5. HRMS (EI) calcd for C30H47N3O8Si (M+) 605.3132, found 605.3135. Anal. Calcd for C30H47N3O8Si: C, 59.48; H, 7.82; N, 6.94; found: C, 59.33; H, 7.94; N, 6.69. N-Acetoxymethyl-N-(N-benzyloxycarbonyl-L-valyl)-4-oxo-L-homoalanyl-4R-(tercbutyldimethyl)-silyloxy-L-proline methyl ester (41): A solution of Cbz-Val-Hyp-Hyp(TBS)-OMe (40) (122.0 mg, 0.2 mmol) in dry dichloromethane (4.0 mL) was treated with DIB (130.0 mg, 0.4 mmol) and iodine (51.0 mg, 0.2 mmol). The reaction mixture was stirred at 26 oC for 3 h, under irradiation with visible light, then underwent the usual work-up and the residue was purified by rotatory chromatography (hexane/EtOAc, 60:40), affording the aldehyde 41 (83.0 mg, 62%), as a yellowish foam: []D = 38.7 (c 0.58, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.09 (s, 6H), 0.89 (s, 9H), 34


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0.91 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 1.952.18 (m, 6H), 2.74 (dd, J = 5.8, 17.6 Hz, 1H), 3.13 (dd, J = 7.8, 17.3 Hz, 1H), 3.51 (dd, J = 3.2, 10.2 Hz, 1H), 3.70 (s, 3H), 3.75 (m, 1H), 4.484.58 (m, 3H), 5.08 (d, J = 12.3 Hz, 1H), 5.12 (d, J = 12.4 Hz, 1H), 5.24 (br d, J = 7.9 Hz, 1H), 5.58 (br d, J = 11.9 Hz, 1H), 5.63 (br d, J = 12.3 Hz, 1H), 5.72 (m, 1H), 7.287.36 (m, 5H), 9.69 (s, 1H).

13

C NMR

(125.7 MHz, CDCl3, 70 oC) C 4.8, 17.2, 17.9, 19.7, 20.6, 25.7, 31.8, 38.1, 44.1, 49.8, 52.2, 55.0, 56.3, 58.4, 67.1, 69.2, 70.6, 128.0, 128.1, 128.5, 136.6, 156.0, 167.8, 169.5, 171.7, 173.4, 197.4. IR (CHCl3) 3343, 2958, 1744, 1726, 1713, 1659, 1650 cm–1. HRMS (ESI) calcd for C32H49N3O10NaSi (M+ + Na) 686.3085, found 686.3094. N-Acetoxymethyl-N-(N-benzyloxycarbonyl-L-valyl)-4-(2S-benzyloxycarbonyl-1-pyrrolidinyl)-Lhomoalanyl-4R-(t-butyldimethyl)silyloxy-L-proline methyl ester (42): A solution of the peptide 41 (67.0 mg, 0.1 mmol) in dry dichloroethane (2.0 mL) was treated with H-Pro-OBn (53.0 mg, 0.14 mmol) and Et3N (0.02 mL, 0.14 mmol). The mixture was stirred for 10 min, then NaBH(OAc)3 (34.0 mg, 0.16 mmol) was added and the stirring continued for 6 h at 26 oC. The reaction mixture was poured into saturated aqueous NaHCO3 and extracted with dichloromethane. The organic layer was washed with brine and then was dried and concentrated as usual. The residue was purified by rotatory chromatography (hexane/EtOAc, 60:40), affording product 42 (71.0 mg, 83%), as a yellowish oil: []D = 50.63 (c 0.27, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.06 (s, 6H), 0.86 (s, 9H), 0.88 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 1.93-2.15 (m, 12H), 2.50-2.82 (m, 3H), 3.08 (m, 1H), 3.38 (br b, 1H), 3.65 (br b, 1H), 3.68 (s, 3H), 3.87 (br dd, J = 4.6, 10.2 Hz, 1H), 4.46-4.50 (m, 3H), 5.02 (d, J = 12.1 Hz, 1H), 5.09 (d, J = 12.4 Hz, 1H), 5.14 (br s, 2H), 5.28 (br b, 1H), 5.41 (m, 1H), 5.55-5.67 (m, 2H), 7.20-7.35 (m, 10H).

13

C NMR (125.7 MHz, CDCl3, 70 oC) C 4.8, 17.3, 18.0, 19.6, 20.8, 23.5,

25.8, 29.5, 32.0, 38.2, 50.3, 52.1, 53.1, 55.2, 56.3, 58.1, 65.7, 66.3, 67.0, 69.1, 70.8, 127.9, 128.0, 128.2, 128.5, 128.6, 136.7, 156.0, 169.8, 170.0, 172.2, 173.8; a (C) signal was not clearly observed. IR (CHCl3) 3328, 1747, 1731, 1659, 1651, 1506 cm–1. HRMS (ESI) calcd for C44H64N4O11NaSi (M+ + Na) 875.4239, found 875.4246.

35



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N-(N-Benzyloxycarbonyl-L-valyl)-4-[5S-(5-(N-methoxycarbonyl)amino-5-(N-(1-O-methyl)-Lleucine)carbonyl)pentyl]amino-L-homoalanyl-4R-(tert-butyldimethyl)silyloxy-L-proline

methyl

ester (43): A solution of aldehyde 41 (67.0 mg, 0.1 mmol) in dry methanol (2.0 mL) was treated with Boc-Lys-Ile-OMe (19) (52.0 mg, 0.14 mmol) and Et3N (0.02 mL, 0.14 mmol), and stirred for 60 min. Then sodium borohydride was added (6.0 mg, 0.16 mmol) and the mixture was warmed to 45 °C for 18 h. After cooling at 26 oC, it was poured into water and extracted with EtOAc. The organic layer was dried and concentrated as usual, and the residue was purified by rotatory chromatography on silica gel (DCM/MeOH, 90:10), affording product 43 (71.0 mg, 75%), as a yellowish oil: 1H NMR (500 MHz, CD3OD, 70 oC): H 0.00 (s, 6H), 0.88 (s, 9H), 0.890.98 (m, 12H), 1.13 (m, 1H), 1.44 (s, 9H), 1.421.48 (m, 3H), 1.601.65 (m, 3H), 1.702.16 (m, 6H), 2.05 (m, 1H), 2.80 (m, 2H), 2.92 (m, 2H), 3.71 (s, 3H), 3.72 (m, 1H), 3.73 (s, 3H), 3.83 (m, 1H), 3.95 (d, J = 7.0 Hz, 1H), 4.08 (br dd, J = 7.8, 7.8 Hz, 1H), 4.42 (d, J = 5.9 Hz, 1H), 4.53 (dd, J = 8.0, 8.1 Hz, 1H), 4.60 (m, 1H), 4.82 (br dd, J = 5.5, 6.7 Hz, 1H), 5.10 (s, 2H), 7.287.38 (m, 5H). 13C NMR (125.7 MHz, CD3OD, 70 oC): C 4.7, 11.6, 16.0, 18.8, 19.4, 19.8, 24.1, 26.2, 26.4, 27.9, 28.8, 30.8, 31.9, 32.7, 38.7, 39.2, 45.5, 49.0, 50.2, 52.4, 52.9, 55.5, 56.9, 58.2, 59.5, 62.3, 67.9, 72.3, 78.2, 128.8, 129.0, 129.5, 138.3, 171.7, 173.5, 174.0, 174.3, 174.8; two (C) signals, corresponding to the carbamates, were not clearly observed. HRMS (ESI) calcd for C47H81N6O12Si (M+ + H) 949.5682, found 949.5687. N-(N-Benzyloxycarbonyl-L-valyl)-4-[5S-(5-(N-methoxycarbonyl)amino-5-(N-(1-O-methyl)-Lleucine)carbonyl)pentyl](methoxycarbonyl)amino-L-homoalanyl-4R-(tert-butyldimethyl) silyloxyL-proline methyl ester (44): The pentapeptide 43 (30.0 mg, 0.03 mmol) was added to saturated

aqueous NaHCO3 (2.0 mL). The solution was cooled to 0 oC and a solution of methyl chloroformate (4.0 µL, 0.05 mmol) in THF (1 mL) was added. The mixture was allowed to reach 26 oC and stirred for 8 h, then was poured into water and extracted with EtOAc. The organic layer was dried and concentrated as usual, affording the peptide 44 (31.0 mg, 97%) as a yellowish foam: []D = 23.4 (c 0.27, CHCl3). 1H NMR (500 MHz, CDCl3, 70 oC): H 0.10 (s, 6H), 0.88 (s, 9H), 0.900.96 (m, 12H), 1.20 (m, 1H), 1.271.37 (m, 2H), 1.44 (s, 9H), 1.45 (m, 1H), 1.461.56 (m, 2H), 1.59 (m, 1H), 36


Page 37 of 43

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1.781.86 (m, 2H), 1.89 (m, 1H), 2.04 (m, 1H), 2.052.15 (m, 2H), 2.21 (m, 1H), 3.18 (m, 2H), 3.29 (br dd, J = 7.4, 7.6 Hz, 2H), 3.64 (m, 1H), 3.67 (s, 3H), 3.74 (s, 6H), 3.75 (m, 1H), 4.054.15 (m, 2H), 4.54 (m, 1H), 4.574.63 (m, 2H), 4.74 (m, 1H), 5.13 (br b, 1H), 5.15 (br s, 2H), 5.46 (br d, J = 7.8 Hz, 1H), 6.86 (br b, 1H), 6.91 (br b, 1H), 7.29 (m, 1H), 7.317.40 (m, 4H). o

13

C NMR (125.7 MHz, CDCl3, 70

C): C 4.8, 11.3, 15.5, 17.9, 19.2, 22.6, 25.3, 25.7, 28.0, 28.4, 31.1, 31.2, 31.9, 38.0, 38.2, 43.7, 47.2,

49.1, 51.9, 52.1, 52.4, 54.9, 55.4, 56.6, 58.0, 60.6, 67.1, 70.9, 79.9, 127.9, 128.0, 128.4, 136.7, 155.7, 156.4, 156.8, 170.2, 171.0, 172.1, 172.2, 172.4. IR (CHCl3) 3309, 2958, 2933, 1742, 1698, 1681, 1660, 1651, 1644, 1537 cm–1. HRMS (ESI) calcd for C49H82N6O14NaSi (M+ + Na) 1029.5556, found 1029.5533. Acknowledgments. This work is dedicated to Prof. Troels Skrydstrup and Prof. William D. Lubell, and it was mainly supported by the Research Program SAF-2013-48399-R, but also by CTQ2009-07109, Plan Estatal de I+D, Ministerio de Economía y Competitividad, Spain, and European Social Funds (FSE). I.R.E. thanks CSIC (Spanish Research Council) for a JAE predoctoral contract.

Supporting Information Available. Reproductions of 1H and 13C NMR spectra of compounds 920, 2227, 2931, 3542, 44 and 45. This material is available free of charge via the Internet at http://pubs.acs.org. References and Footnotes 1) (a) Jamieson, A. G.; Boutard, N.; Sabatino, D.; Lubell, W. D. Chem. Biol. Drug. Des. 2013, 81, 148–165. (b) Dumas, A.; Lercher, L.; Spicer, C. D.; Davis, B. G. Chem. Sci. 2015, 6, 50–69. (c) For other reviews on the subjetc, see: Schumacher, D.; Hackenberger, C. P. R. Curr. Opin. Chem. Biol. 2014, 22, 62–69. (d) Khashper, A.; Lubell, W. D. Org. Biomol. Chem. 2014, 12, 5052– 5070.(e) Walsh, C. T.; O’Brien, R. V.; Khosla, C. Angew. Chem. Int. Ed. 2013, 52, 7098–7124. (f) Takaoka, Y.; Ojida, A.; Hamachi, I. Angew. Chem. Int. Ed. 2013, 52, 4088–4106. (g) Pasut, G.; Veronese, F. M. J. Control. Release 2012, 161, 461–472. (h) Díaz-Rodríguez, A.; Davis, B. G. 37



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Domino Process Achieves Site-Selective Peptide Modification with

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