Photocyclization of Tetra- and Pentapeptides Containing

Article Cite This: J. Org. Chem. 2018, 83, 14905−14922

pubs.acs.org/joc

Photocyclization of Tetra- and Pentapeptides Containing Adamantylphthalimide and Phenylalanines: Reaction Efficiency and Diastereoselectivity Margareta Sohora, Mario Vazdar,* Irena Sović, Kata Mlinarić-Majerski, and Nikola Basarić* Department of Organic Chemistry and Biochemistry, Rud̵er Bošković Institute, Bijenička cesta 54, 10000 Zagreb, Croatia

Downloaded via YORK UNIV on December 21, 2018 at 11:25:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: A series of tetrapeptides and pentapeptides was synthesized bearing a phthalimide chromophore at the Nterminus. The C-terminus of the peptides was strategically substituted with an amino acid, Phe, Phe(OMe), or Phe(OMe)2 characterized by different oxidation potentials. The photochemical reactivity of the peptides was investigated by preparative irradiation and isolation of photoproducts, as well as with laser flash photolysis. Upon photoexcitation, the peptides undergo photoinduced electron transfer (PET) and decarboxylation, followed by diastereoselective cyclization with the retention of configuration for tetrapeptides or inversion of configuration for pentapeptides. Molecular dynamics (MD) simulations and NOE experiments enabled assignment of the stereochemistry of the cyclic peptides. MD simulations of the linear peptides disclosed conformational reasons for the observed diastereoselectivity, being due to the peptide backbone spatial orientation imposed by the Phe amino acids. The photochemical efficiency for the decarboxylation and cyclization is not dependent on the peptide length, but it depends on the oxidation potential of the amino acid at the C-terminus. The results described herein are particularly important for the rational design of efficient photochemical reactions for the preparation of cyclic peptides with the desired selectivity.

■

INTRODUCTION A large number of synthetic and natural peptides is characterized by biological activity.1 However, peptides are not often considered as drugs, primarily due to facile degradation by proteolytic enzymes and low probability to cross cell membranes. On the contrary, cyclic peptides are more appealing as potential leads,2 primarily due to their higher stability toward hydrolysis and specific 3D shapes3 which allows them to interact with different receptors,4 particularly with proteins. Moreover, it has been shown that cyclic peptides penetrate cell membranes more easily than linear analogues.5 Thus, the bactericidal, immunosuppressive, antiangiogenic,5 antiviral,6 cytotoxic, and anticancer activities7,8 of cyclic peptides have been demonstrated and reviewed.9 Consequently, there has been a significant increase in the number of cyclic peptides that are taken orally as drugs.10 Synthetic methods for preparation of cyclic peptides and peptidomimetics have been based on the usual peptide coupling protocols by activation of the carboxylic functional group in solution or on a solid support.11 In particular, amide © 2018 American Chemical Society

bond formation via side-chain cyclization, cyclization via an orthogonal coupling strategy, enzyme-catalyzed cyclization, and macrocyclizations using functions other than the main peptide backbone have been used.12 More recent modern examples for the macrocyclization involve metal-ion-assisted cyclizations, sulfur-mediated cyclizations, ring-contraction strategies involving lactones, azide−alkyne cycloadditions, ring-closing metathesis, use of isocyanides and multicomponent reactions, or electrostatically controlled macrocyclizations.13 Photochemical methods are also interesting in the synthetic strategy toward peptidomimetics. Mariano et al. developed photoinduced cyclization initiated by single electron transfer (SET) promoted desilylation,14 whereas Mariano et al.14 and Griesbeck et al.15 used a photoinduced decarboxylation protocol. The photodecarboxylation was initiated by SET from the carboxylic acid to phthalimide in the triplet excited state, and it was demonstrated that the yields of cyclic Received: July 13, 2018 Published: November 21, 2018 14905

DOI: 10.1021/acs.joc.8b01785 J. Org. Chem. 2018, 83, 14905−14922

Article

The Journal of Organic Chemistry peptides depended on the peptide conformation.16 Furthermore, Mariano et al. proposed that the efficiency of the photocyclization depended on the relative stabilities and equilibration of the peptide intrastrand electron transfer states.17 We have become interested in the photoinduced phthalimide-promoted preparation of polycyclic molecules18−20 and peptide cyclizations21 and found that photocyclization of adamantane dipeptides proceeds enantioselectively with a memory of chirality.22 However, when we tried to expand the use of the photodecarboxylation protocol to peptides containing tyrosine at the peptide C-terminus, we found that the photodecarboxylation did not take place.23 Moreover, the lack of photocatalytic decarboxylation reactions of tyrosinecontaining peptides initiated by phenanthrene and dicyanobenzene has been demonstrated by Yoshimi et al., indicating that the C-terminus affects the subsequent reactions after the initial SET.24 Herein, we report on a more general study of the tetra- and pentapeptide cyclizations 1−6 by a photodecarboxylation protocol where we systematically changed the peptide Cterminus and investigated its effects on the cyclization efficiency. The C-terminus is modified by a different number of methoxy groups at the Phe to change its oxidation potential, whereas the chain length is anticipated to affect the rate of SET. Moreover, we found that photocyclization takes place diastereoselectively. The reasons for the observed selectivity were fully disclosed by molecular modeling.

tyrosine derivatives.23 These dipeptides or tripeptides were then coupled with the NHS-activated dipeptide 22 to afford target molecules 1−6. The succinimide activation protocol gave low yields on pentapeptides, particularly on 6. Therefore, some target peptides were also prepared from Bn-protected amino acids by use of a stronger coupling reagent, N,N,N′,N′-tetramethylO-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBT).26 Bn-protected compounds were synthesized as shown for the noncanonical amino acid (Scheme 3), which was first transformed to Boc derivative 23, and then the Bn group was introduced. The Boc group was cleaved off from 24 to afford Bn-proteced amino acid 25. In an analogous way, HPhe-OBn was prepared and used for the preparation of PhePhe dipeptide 27 (Scheme 4). The HBTU coupling protocol was applied in the synthesis of tripeptides 28 and 30, starting from Boc-Phe-Phe-OH 12 (Scheme 5). The Boc group from the OBn-protected tripeptides was cleaved off, yielding 29 and 31, which were used in the final peptide coupling reactions. To synthesize the target molecules, the carboxylic acid in dipeptide 21 was activated by HBTU and treated with Bnprotected peptides 27, 29, and 31. The Bn group was cleaved off, as shown in the example of pentapeptide 32 in Scheme 2, by use of Et3SiH and Pd/C, which was found as a protocol compatible with the phthalimide group.21 Photochemistry. On the basis of literature precedent,14−16,21,22 irradiation of peptides is anticipated to give two types of products, cyclic peptides 33−38 and simple decarboxylation products 39−44 (Scheme 6). To isolate anticipated photoproducts, we conducted preparative irradiations of peptides 1−6. Selective excitation of the phthalimide moiety in peptides was accomplished by use of lamps with a maximal output at 300 nm, where the phthalimide absorbs light, and Phe, Phe(OMe), or Phe(OMe)2 do not absorb light (for the absorption spectra see Figures S1 and S2 in the SI). The irradiations were performed in CH3CN−H2O in the presence of K2CO3 as a base which is required to deprotonate the carboxylic acid at the C-terminus, namely, phthalimide derivatives in the triplet excited state react in photoinduced SET with reducing agents that have the donor oxidation potential < 1.6 V vs SCE.27 Since carboxylates are better reducing agents than carboxylic acids,28−30 deprotonation is required for the SET to occur. Irradiations were also conducted in acetone−H2O in the presence of K2CO3 where upon irradiation at 300 nm acetone is excited and acts as a triplet sensitizer. The efficient exergonic energy transfer is anticipated only to phthalimide or Phe(OMe)2 and not to Phe or Phe(OMe) (ET acetone = 332 kJ mol−1, ET phthalimide = 297 kJ mol−1, ET toluene = 347 kJ mol−1, ET phenol = 342 kJ mol−1, for calculated energies see Table S2 in the SI).31 NMR yields of cyclic peptides after the irradiations are compiled in Table 1. The photochemical reaction proceeds more efficiently upon sensitized irradiation. Furthermore, in some cases photocyclization was not clean, and numerous decomposition and/or high molecular weight products were formed. Attempts to isolate these side products failed. However, the photocyclization proceeds selectively, giving only 33−38 if it is conducted under acetone sensitization in high dilution in the presence of a buffer and to low conversion (30−40%). Photocyclization is anticipated to give simple decarboxylation side products 39−44. However, NMR spectra after photolyses indicated the presence of starting acyclic peptides,

■

RESULTS Synthesis. Synthesis of peptides 1 and 2 was conducted in solution and on a solid support, whereas 3−6 were prepared in solution only. In the solid-supported synthesis, Wang resin modified with Phe and the standard Fmoc protective group chemistry were used. The amino acids, Phe or phthalimidoadamantane carboxylic acid, were activated by use of HBTU, and cleavage of the Fmoc was accomplished by use of piperidine. After synthesis of tetra- or pentapeptides, cleavage from the resin by TFA afforded peptides 1 and 2 that were purified by chromatography and isolated in 33% and 29% yield, respectively. The general procedure for the solution-phase synthesis is shown in Schemes 1, 2, 3, 4, and 5. Synthesis of dipeptides 10 and 11 and tripeptides 19 and 20 containing noncanonical amino acids at the C-terminus was conducted by the usual Nhydroxysuccinimide (NHS) and N,N′-dicyclohexylcarbodiimide (DCC) activation,25 as we recently described for the 14906


Article

The Journal of Organic Chemistry Scheme 1. Synthesis of Di- and Tripeptide Intermediates 8−20

Scheme 2. Synthesis of Tetra- and Pentapeptides 1−6

Scheme 3. Synthesis of Phe(OMe)2 Intermediate 25

14907


Article

The Journal of Organic Chemistry Scheme 4. Synthesis of PhePhe Dipeptide Intermediate 27

Table 1. Photoproduct Yields in the Photochemistry of Peptides 1−6a starting peptide/solvent irradiation time/conversion % 1/CH3CN−H2Ob 1/acetone−H2Oc 2/CH3CN−H2Ob 2/acetone−H2Oc 3/CH3CN−H2Ob 3/acetone−H2Ob 4/CH3CN−H2Ob 4/acetone−H2Oc 5/CH3CN−H2Ob 5/acetone−H2Oc 6/acetone−H2Ob

Scheme 5. Synthesis of Tripeptide Intermediates 29 and 31

40 10 30 10 20 20 30 20 10 15 30

cyclic peptide (%)

min/49 min/35 min/12 min/56 min/67 min/95 min/45 min/95 min/5 min/56 min/40

33 33 34 34 35 35 36 36 37 37 38

(41) (20) (5) (34) (51) (59) (28) (42) (0) (13) (24)

a

Irradiated in the presence of 0.5 equiv of K2CO3 with 8 lamps at 300 nm (1 lamp = 8 W). The yields were determined from NMR spectra of the crude mixture after photolysis. bIrradiated volume = 15 mL; c = 5 × 10−4 M. cIrradiated volume = 50−75 mL; c = 1 × 10−3 M.

Scheme 7. Synthesis of Intermediates 46 and 48

cyclic peptides, and decomposition material. Moreover, chromatographic separations enabled recovery of the starting peptide and isolation of cyclic peptides, and only from peptide 5 was simple decarboxylation product 43 isolated. To verify if decarobxylation products were formed, 39 and 40 were synthesized by the HBTU peptide coupling protocol (Schemes 7 and 8). Boc-Phe-OH or Boc-Phe-Phe-OH was coupled with phenylethylamine to afford dipeptide 45 and tripeptide 47. The Boc group was cleaved off to give TFA salts 46 and 48, which were used in the coupling with 21 to afford the anticipated decarboxylation products 39 and 40. HPLC analysis of the crude mixture after irradiation of peptides 1 and 2 and authentic samples of 39 and 40 synthesized by an

independent method indicated that 39 and 40 were formed in the photochemical reaction but only in trace amounts ( 7 is not clear. To compare the relative reactivity of peptides 1−6, quantum yields for the photocyclization (ΦR) were determined. The efficiency under direct excitation was determined by use of a KI/KIO3 actinometer (Φ254 = 0.74).31,32 The peptides were irradiated in CH3CN−H2O (1:2) at 254 nm in the presence of potassium phosphate buffer (c = 50 mM, pH = 6.0). For the sensitization conditions, efficiencies were determined by use of 4-[(N-phthalimido)methyl]cyclohexane carboxylic acid (Φ = 0.30) as a secondary actinometer (Scheme S1 in the SI).33 Irradiations were performed at 300 nm in acetone−H2O (1:1) in the presence of potassium phosphate buffer (c = 50 mM, pH = 6.0). Results are compiled in Table 2, whereas details on the determination of quantum yields can be found in the SI. The ΦR for the photocyclization in CH3CN upon excitation at 254 nm are 5−25 times lower than in the acetone-sensitized conditions, and there is no clear trend on ΦR depending on the molecular structure. Since peptides are multichromophoric molecules, the values of ΦR depend on the probability to excite the phthalimide chromophore at 254 nm. Phthalimide, Phe(OMe), and Phe(OMe)2 have similar absorptivities at 254 nm, whereas Phe has about five times lower molar absorption coefficient (Figure S2 in the SI). Therefore, 1 and 2 react more efficiently upon excitation at 254 nm than peptides 3−6. On the contrary, there is a clear trend for ΦR in the acetone-sensitized reaction. In this experiment, acetone was

Figure 1. Structure of cyclic tetrapeptide (S,S)-33 and pentapeptide (S,S,R)-34 and important NOE interactions.

Note the different stereochemistry in tetrapeptides and pentapeptides. The stereogenic center at the C atom where decarboxylation took place in pentapeptides underwent a complete inversion of the configuration, whereas in tetrapeptides the stereochemistry was retained. Besides NOESY experiments, a different configuration in tetra- and pentapeptides was also indicated from their CD spectra where bands with the opposite sign were observed for tetra- and pentapeptides (see Figures S10−S12 in the SI). The observed stereoselectivity may be due to an epimerization reaction taking place under basic conditions after the photochemical cyclization step. In principle, under basic conditions formation of enolate could lead to epimerization and give a more stable cyclic peptide. To investigate if the epimerization in cyclic peptides takes place, we calculated free energies for some typical conformers of diastereomers (S,S)-33 and (S,R)-33 as well as for pentapeptides (S,S,R)-34 and (S,S,S)-34. The calculated free energies of diastereomers at the SMD(acetone)/B3LYP/6-311+G(d,p)// B3LYP/6-31G(d) level of theory (Table S1 in the SI) indicate that we have isolated less stable tetrapeptide isomer (S,S)-33, whereas pentapeptides have similar free energies. Thus, if the epimerization took place, we would mainly obtain isomer (S,R)-33, whereas diastereomers (S,S,R)-34 and (S,S,S)-34 would be formed in equal amounts. We performed additional experiments where cyclic peptides 33 and 34 were dissolved in d6-acetone−D2O and treated with 0.5 equiv of K2CO3 to simulate basic conditions in the photochemical reaction. 1H NMR and HPLC-MS analyses over several days did not indicate the epimerization or incorporation of deuterium at the 14909


Article

The Journal of Organic Chemistry Table 2. Quantum Yields for the Phodecarboxylative Cyclization (ΦR) of Peptides 1−6a compound 1 2 3 4 5 6

ΦR (CH3CN−H2O)b 0.050 0.057 0.014 0.027 0.020 0.008

± ± ± ± ± ±

0.005 0.007 0.003 0.003 0.006 0.002

ΦR (acetone−H2O)c 0.27 0.29 0.36 0.39 0.08 0.09

± ± ± ± ± ±

0.02 0.04 0.03 0.04 0.02 0.02

a

Measurements were done in triplicate, and the average value is reported. The quoted error corresponds to the maximum absolute deviations. bMeasurement were conducted by irradiating at 254 nm CH3CN−H2O (1:2) by use of a KI/KIO3 actinometer (Φ254 = 0.74).31,32 cMeasurements were conducted by irradiating at 300 nm in acetone−H2O (1:1) by use of a secondary actinometer; photoreaction of 4-[(N-phthalimido)methyl]cyclohexane carboxylic acid (Φ = 0.30).33

excited only, and the energy transfer to the phthalimide took place. The same concentration of actinometer (phthalimide derivative) and peptides allowed for the approximation that the energy transfer from acetone to the actinometer and peptides took place with the same efficiency. ΦR values do not depend on the peptide chain length, but they depend on the oxidation potential of the C-terminus amino acid. Thus, ΦR is the highest for Phe(OMe) derivatives 3 and 4 and the lowest for peptides 5 and 6. Molecular Dynamics Simulations. In order to verify the assigned stereochemistry of the cyclic peptides 33 and 34 based on the NOESY spectra, we performed molecular dynamics (MD) simulations and checked for the close contacts between the selected H atoms (Figure 1) in two possible tetrapeptide diastereomers with the (S,S) and (S,R) configurations as well as pentapeptide with the (S,S,R) and (S,S,S) configurations, respectively. The dynamic properties were based on 500 ns of MD simulations in the corresponding NMR solvent (see the SI). Figure 2 shows two typical snapshots for two diastereomers of the cyclic tetrapeptide 33one with the S,S and the other with the S,R configuration. Analysis of the distances dS,S and dS,R between two H atoms corresponding to those with the NOE interactions (Figure 1) clearly indicates that the cyclic tetrapeptide 33 with the S,S configuration is dominant in MD simulations, thus confirming the assigned structure, namely, the distance dS,S assumes values of ca. 3.0−3.5 Å, which is in agreement with the NOE signal observed. On the contrary, the distances dS,R in the S,R diastereomer are longer on average and cannot contribute to the NOE signal. On the basis of the simulation data, a similar conclusion can be drawn for the cyclic pentapeptide 34. In particular, the distances dS,S,R in the S,S,R configuration are in the range of 2− 3 Å (Figure 3), which strongly contribute to the NOE signal observed. The distances dS,S,S are on average longer and cannot give rise to the NOE signal. Consequently, MD simulations confirm the assigned structure of pentapeptide with the inversion of configuration at the chiral C atom where decarboxylation took place. To get information on the conformational flexibility of peptides and the possibility that those two reactive carbon atoms in the photocyclization reaction approach close to each other, MD simulations were performed on potassium salts of linear tetrapeptide 1 and pentapeptide 2 with the Sconfigurations at the chiral centers. In particular, we analyzed

Figure 2. Snapshots from MD simulations for cyclic tetrapeptide 33 in the S,S and the S,R configuration with indicated H atoms that are in the NOE interaction, and corresponding distances dS,S and dS,R between them (top). Histogramic representation of distances dS,S and dS,R during the simulation time (bottom).

Figure 3. Snapshots from MD simulations for cyclic pentapeptide 34 in the S,S,S and S,S,R configuration with indicated H atoms that are in the NOE interaction, and corresponding distances dS,S,R and dS,S,S between them (top). Histogramic representation of distances dS,S,S and dS,S,R during the simulation time (bottom).

14910


Article

The Journal of Organic Chemistry the distance between the reactive C atom in the phtalimide moiety and the reactive C atom in the peptide backbone in 1 and 2 (Figure 4). The analysis shows that for both peptides 1

Table 3. LFP Data for Peptides 1−5 comp./solvent 1/CH3CN 1/CH3CN−H2O (K2CO3) 2/CH3CN 3/CH3CN 4/CH3CN 5/CH3CN

ΦISCb

kq (dm3 mol−1s−1)c

1.6 ± 0.1 2.2 ± 0.1

0.05−0.06

(1.6 ± 0.1) × 109 (5.6 ± 0.1) × 108

1.65 ± 0.05 0.6 ± 0.1 1.0 ± 0.1 0.6 ± 0.1

0.05−0.06

(1.6 ± 0.1) × 109

τ (μs)a

a

Lifetime of the transient in N2-purged solution. bQuantum yield of ISC for the CH3CN solution; determined by comparing the intensity of the signal with the optically matched solution of Nmethylphthalimide in CH3CN at 266 nm (ΦISC = 0.8).35 cRate constant for the quenching with O2.

Figure 5. Transient absorption spectra of tetrapeptides 1, 3, and 5 in N2-purged CH3CN.

Figure 4. Snapshots from MD simulations for tetrapeptide 1 and pentapeptide 2 with indicated reactive C atoms, and corresponding distances d1 and d2 between them (top). Histogramic representation of distances d1 and d2 during the simulation time (bottom).

crossing (ISC) for 1 was estimated by comparison of the intensity of the transient absorption immediately after the laser pulse with the optically matched solution of N-methylphthalimide (ΦISC = 0.8).35 The ΦISC represents only a lower limit since 266 nm light can also excite Phe. However, absorptivity of phthalimide at 266 nm is six times higher than Phe (Figure S2 in the SI). Compared to simple N-alkylphthalimide, the ISC in 1 is fairly inefficient, which is probably the reason for the observed low ΦR for the formation of cyclic peptide upon irradiation in CH3CN−H2O. Low ΦISC for N-adamantylphthalimides has been reported.19 In basic aqueous solution, a photoinduced SET is expected to take place, giving rise to phthalimide radical−anion. The photoinduced SET probably takes place in the triplet excited state. However, SET in the singlet excited state cannot be completely ruled out. Very short singlet excited state lifetimes of N-alkylphthalimides are known (N-methylphthalimide τ = 185 ps),36 and they may account for the low ΦR observed upon 266 nm excitation. However, in basic aqueous solution the transient absorption spectrum had the same appearance as in the neat CH3CN no additional band was observed. In basic aqueous solution, a transient was detected with the maximum of absorption at 330 nm and lifetime of 2.2 ± 0.1 μs (Figures S14 and S15 in the SI). It was also quenched by O2, so it was assigned to the phthalimide triplet excited state. The phthalimide radical−anion was not detected. Transient absorption spectra of 2 are similar to those of 1. However, 3−5 gave rise to different transients (Figure 5). The absorption maxima for 3 and 4 were observed at 400 nm and a shoulder at 500 nm, whereas the phthalimide triplet was not detected. On the basis of literature precedent the maximum at

and 2 the distance between the reactive C atoms quite often approaches the value of ∼5 Å, which is a sufficient prerequisite for efficient photocyclization after PET-promoted photodecarboxylation and subsequent intersystem crossing of the triplet biradical to the singlet manifold.34 Moreover, the difference in the dynamic behavior and conformational flexibility is similar for both tetrapeptide 1 and pentapeptide 2. However, an additional Phe moiety in 2 introduces a turn in the molecule and different spatial orientation of the groups attached to the chiral C atom where the photocyclization takes place (Figure 4). The different spatial orientation ultimately leads to the inversion of chirality in the cyclic pentapeptide 34 in contrast to the cyclic tetrapeptide 33. Laser Flash Photolysis (LFP). To detect triplet excited states and other plausible transients in the photocyclization of peptides, LFP was conducted. The samples were excited with a YAG laser at 266 nm, and the measurements were conducted in N2- and O2-purged solutions where the difference due to O2 quenching of the triplet excited states and radicals was anticipated. Data obtained by LFP are summarized in Table 3 and shown in Figures S14−S17 in the SI. In the N2-purged CH3CN solution of 1 we detected a transient with the absorption maximum at 330 nm that decayed with unimolecular kinetics and lifetime of 1.6 ± 0.1 μs (Figure 5 and Figure S14 in the SI). It was assigned to the phthalimide triplet state, based on the comparison with the published spectra for N-alkyl phthalimides and quenching with O2.35 Furthermore, the quantum yield for the intersystem 14911


Article

The Journal of Organic Chemistry Scheme 9. Mechanism for Photochemical Cyclization of Peptides 3 and 5

400 nm can be assigned to methoxybenzene radical−cation37 and the shoulder at higher wavelength to phthalimide radical− anion19,33,35 formed by photoinduced SET.

PET (quantum yield > 0.8−0.9) followed by efficient irreversible decarboxylation and cyclization. However, the reaction efficiency is highly dependent on the solution pH, since it is important that the carboxylate is deprotonated. Highly efficient electron transfer38 after inefficient ISC in the N-adamantylphthalimide photochemistry19 has been documented. On the other hand, the PET in 3−6 probably takes place between the Phe(OMe) or Phe(OMe)2 and the phthalimide, giving rise to RIP-1. The methoxybenzene or dimethoxybenzene radical−cation and the phthalimide radical−anion were detected by LFP. For the cyclization reaction, the second intrastrand ET between the radical−cation and the carboxylate should take place, leading to RIP-2 and followed by an irreversible decarboxylation (Scheme 9). Since the dimethoxybenzene radical−cation is more stable than the methoxybenzene radical−cation, the intrastrand ET is more favorable in 3 and 4 than in 5 and 6, namely, introduction of an additional methoxy group decreases the oxidation potential of the electron acceptor in RIP-1 (radical−cation) and hence increases the barrier for intrastrand ET. Consequently, compounds bearing one methoxy group undergo the most efficient cyclizations. The other plausible explanation why compounds 5 and 6 undergo less efficient cyclization under acetone sensitization than 3 and 4, respectively, may be due to competitive triplet energy transfer from acetone to phthalimide and dimethoxytoluene moiety. The calculated triplet excited state energy of dimethoxytoluene (ET = 328 kJ mol−1 Table S2 in the SI) is similar to the reported triplet energy for acetone. However, the energy transfer from acetone to dimethoxytoluene is probably less efficient than to the phthalimide. Furthermore, if the Phe(OMe)2 triplet was populated it would also undergo PET from the phthalimide in the ground state resulting in the same RIP-1.

■

DISCUSSION Understanding the reactivity in the peptide cyclization reactions promoted by photoexcited phthalimide is important in the design of new reactive systems and applications in organic synthesis. Here we discuss two important aspects concerned with the correlation of molecular structure and reactivity and the stereoselectivity of the cyclization. The photocyclization reaction efficiency is directly correlated with the photochemical reactivity. Thus, although the absolute values for the rates of photoinduced electron transfer (PET) and the photocyclization were not determined, measurements of the reaction quantum yields gave inside in the overall reactivity that can be correlated with the molecular structure. Upon direct photoexcitation at 254 nm efficiency in photochemical reaction is not only correlated to the intrinsic reactivities of the molecules but also to the absorptivities of the chromophores at that wavelength. However, there is a trend in the reactivity with molecular structure upon sensitization with acetone. Molecules bearing one methoxy group are the most reactive, whereas the distance of the peptide backbone between the electron donor (carboxylate) and the acceptor (phthalimide) does not affect the reaction efficiency. The result suggests that the slowest step in the photocyclization is the PET which is probably taking place through space in the conformer where the phthalimide and the peptide C-terminus are close. However, the PET in 1 and 2 probably takes place between the carboxylate and the phthalimide. Similar efficiencies for ISC (Table 3) and cyclization quantum yields (Table 2) suggest that the slowest step for 1 and 2 is ISC. Once the phthalimide triplet is populated it undergoes efficient 14912


Article

The Journal of Organic Chemistry

tetramethylsilane as a reference. Silica gel or alumina were used for chromatographic purification. Solvents were purified by distillation. The chemicals for synthesis were obtained from the usual commercial sources. Photochemical reactions were carried in a reactor in quartz test tubes or in a quartz Erlenmeyer flask. Acetone of p.a. purity used in the irradiation experiments was additionally purified by distillation in the presence of KMnO4, whereas CH3CN was of HPLC purity. For the HRMS analyses, the samples were analyzed in a positive mode applying nanoUPLC-ESI-qTOF on instruments. The known precursors, 2-{[3-(N-phthalimido)adamantan-1-yl]carboxamido}acetic acid (21)38 and benzyl-2-{[3-(N-phthalimido)adamantan-1yl]carboxamido}acetate (19), were prepared according to the literature precedent.21 General Procedure for the Solid-Supported Synthesis of 1 and 2.40 Wang resin (100 mg, ∼0.08 mmol) modified with PheFmoc (0.5−0.8 mmol/g, 200−400 Mesh) was treated with DMF (3 mL) to swell and the next day washed with some fresh DMF. After removal of the solvent, 20% piperidine in DMF (3 mL) was added, the resin was treated for 2 min, and the solvent was removed. The procedure was repeated again whereupon piperidine in DMF was allowed to react with the resin for 20 min. After completion of the reaction, the resin was washed with DMF (3 × 3 mL). In a separate flask, an amino acid (0.4 mmol), HBTU (151.7 mg, 0.4 mmol), HOBT (61.2 mg, 0.45 mmol), DIPEA (140 μL, 0.8 mmol), and DMF (3 mL) were stirred for 5 min, and the mixture was added to the resin and allowed for the coupling to take place over 20 min. After the coupling was completed, the resin was washed with DMF (3 × 3 mL). The procedure including the Fmoc deprotection, the amino acid activation, and the peptide coupling was repeated until the desired peptide was made. Peptides 1 and 2 were cleaved off from the resin after washing with DMF (3 × 2 mL), CH2Cl2 (3 × 2 mL), and CH3OH (2 × 2 mL) by treatment with 95% TFA−H2O (3 mL) over 40 min. The solution after cleavage was transferred to a cuvette, and diethyl ether (5 mL) was added. The mixture was centrifuged, and the solid peptides were separated from the solution by use of a pipet. The solid peptides were washed with CH3OH (3 mL), DMF (3 mL), CH2Cl2 (3 mL), and hexane (3 mL) and dried at 40 °C and 10 mbar to afford 1 (18 mg, 33%) and 2 (19 mg, 29%). The peptides were additionally purified on a column of silica gel by use of 0−10% CH3OH/CH2Cl2 as eluent. PhthAd-Gly-Phe-Phe-OH (1). Colorless solid; mp 128−130 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.76−7.71 (m, 4H), 7.25−7.14 (m, 10H), 4.63 (ddd, J = 8.2, 5.4, 2.8 Hz, 2H), 3.80 (d, J = 16.2 Hz, 1H), 3.70 (d, J = 16.2 Hz, 1H), 3.19 (dd, J = 14.1, 5.4 Hz, 1H), 3.10 (dd, J = 14.1, 5.4 Hz, 1H), 3.02 (dd, J = 13.9, 8.4 Hz, 1H), 2.87 (dd, J = 13.9, 8.4 Hz, 1H), 2.60 (d, J = 12.9 Hz, 2H), 2.57−2.53 (m, 2H), 2.46 (d, J = 12.3 Hz, 2H), 2.27 (s, 2H), 1.89 (d, J = 12.4 Hz, 2H), 1.82−1.79 (m, 3H), 1.70 (d, J = 12.6 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ/ppm 179.8 (s, 1C), 174.2 (s, 1C), 173.0 (s, 1C), 171.4 (s, 2C), 170.9 (s, 1C), 138.3 (s, 1C), 138.0 (s, 1C), 135.2 (d, 2C), 133.1 (s, 2C), 130.4 (d, 2C), 130.3 (d, 2C), 129.5 (d, 2C), 129.4 (d, 2C), 127.8 (d, 2C), 123.5 (d, 2C), 61.4 (s, 1C), 55.4 (d, 1C), 55.1 (d, 1C), 44.1 (s, 1C), 43.8 (t, 1C), 42.3 (t, 1C), 40.4 (t, 2C), 38.9 (t, 2C), 38.7 (t, 1C), 38.4 (t, 1C), 36.3 (t, 1C), 31.0 (d, 2C); IR (KBr) νmax/cm−1 3393, 3322, 3065, 3029, 2916, 2856, 1715, 1520, 1453, 1367, 1314, 1116, 1078, 719, 701, 641. HRMS (nano UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C39H40N4O7 677.2975; Found 677.2983. [α]D20 +6.06 (c 0.495, 10% CH3OH/CH2Cl2). PhthAd-Gly-Phe-Phe-Phe-OH (2). Colorless solid; mp 151−153 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.73−7.70 (m, 4H), 7.25−7.07 (m, 15 H), 4.65−4.60 (m, 2H), 4.57 (dd, J = 8.5, 5.1 Hz, 1H), 3.78 (d, J = 18.0 Hz, 1H), 3.70 (d, J = 18.0 Hz, 1H), 3.18−3.09 (m, 2H), 3.02−2.96 (m, 2H), 2.90 (dd, J = 14.0, 9.0 Hz, 1H), 2.82− 2.77 (m, 1H), 2.58−2.51 (m, 4H), 2.42 (d, J = 12.0 Hz, 2H), 2.23 (s, 2H), 1.86 (d, J = 12.3 Hz, 2H), 1.82−1.74 (m, 3H), 1.67 (d, J = 12.6 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ/ppm 179.8 (s, 1C), 174.1 (s, 1C), 173.0 (s, 1C), 172.9 (s, 2C), 171.6 (s, 1C), 170.9 (s, 1C), 138.4 (s, 1C), 138.2 (s, 1C), 138.0 (s, 1C), 135.2 (d, 2C), 133.0 (s, 2C), 130.4 (d, 2C), 130.32 (d, 2C), 130.28 (d, 2C), 129.5 (d, 2C), 129.45 (d, 2C), 129.4 (d, 2C), 127.8 (d, 1C), 127.75 (d, 1C), 127.6

Stereochemical aspects of the peptide cyclization reaction are at first glance counterintuitive. Since the decarboxylation creates a radical center, one would expect nonselective radical cyclization leading to a diastereomeric mixture of cyclic peptides. However, Griesbeck39 and we22 have reported phthalimide-induced cyclizations with a memory of chirality, where the axial chirality of the molecule allowed for the enantioselective formation of products. In the cyclizations of peptides 1−6, a similar axial chirality imposed by the adamantane and the phthalimide moiety is preserved. In addition, the peptide backbone contains chiral centers and the Phe moieties induce peptide turns, resulting in a helical chirality. Therefore, cyclization of biradical 3BR proceeds highly diastereoselectively. Furthermore, the cyclization of tetrapeptides proceeds with the retention of the configuration, whereas in pentapeptides the configuration is inverted. The plausible reasons for the observed difference in stereochemistry in cyclic peptides can be obtained by inspection of the tetraand pentapeptide biradical conformations (see Figure 4). Although a large number of the reactive conformations exist where the reactive centers are close, in most of them there is the stabilization by ππ interaction between the phthalimide and the phenyl ring of the peptide C-terminus for both tetraand pentapeptides. This ππ interaction and an additional Phe moiety in the pentapeptide enforces the reactive C atom in 3BR to turn away and adopts a conformation which upon cyclization results in the inversion of the configuration on that atom. Such a driving force in tetrapeptides does not exist, allowing for the cyclization with retention of the configuration. However, due to the large number of conformations, it is not possible to give accurate energy estimates between the conformers that would give rise to two diastereomers. Therefore, it is highly plausible that the most stable conformer of the triplet biradical 3BR undergoes ISC followed by irreversible cyclization to cycl-1. Consequently, the intrinsic chirality and spatial orientation of the peptides and turns imposed by Phe drive the cyclization highly diastereoselectively with the retention of the configuration for tetra- and inversion of the configuration for pentapeptides.

■

CONCLUSIONS Investigation of the photochemical reactivity of a series of newly synthesized tetra- and pentapeptides bearing the phthalimde chromophore at the N-terminus is disclosed. Our main findings are that the photocyclization efficiency does not depend on the peptide chain length, but it depends on the oxidation potential of the amino acid at the C-terminus. For the efficient cyclization, the oxidation potential of the Cterminal amino acid has to be finely tuned to allow fast PET but also to allow the intrastrand ET leading to the irreversible decarboxylation. Moreover, we found that the spatial orientation of the peptide backbone imposed by the Phe turns leads to different stereochemistry in the cyclic tetra- and pentapeptides with the retention or inversion of the configuration, respectively. The results are particularly important for the rational design of new photochemical reactions for the preparation of cyclic peptides with the desired stereoselectivity.

■

EXPERIMENTAL SECTION

General. 1H and 13C NMR spectra were recorded on a spectrometer at 300 or 600 MHz. All NMR spectra were measured in CDCl3, DMSO-d6, CD3OD, CD3CN, or CD3COCD3 using 14913


Article


(s, 1C), 173.0 (s, 1C), 172.4 (s, 1C), 170.8 (s, 2C), 159.8 (s, 1C), 138.6 (s, 1C/2C), 137.8 (s, 1C/2C), 135.2 (d, 2C), 133.0 (s, 1C), 131.5 (d, 2C), 130.6 (s, 1C), 130.2 (d, 4C), 129.6 (d, 2C), 129.4 (d, 2C), 127.8 (d, 1C), 127.7 (d, 1C), 123.5 (d, 2C), 114.8 (d, 2C), 61.3 (s, 1C), 56.5 (d, 1C), 56.3 (d, 1C), 56.2 (d, 1C), 55.6 (q, 1C), 44.2 (t, 1C), 44.0 (s, 1C), 42.1 (t, 1C), 40.2 (t, 2C), 38.79 (t, 1C), 38.74 (t, 1C), 38.4 (t, 1C), 38.0 (t, 1C), 37.9 (t, 1C), 36.2 (t, 1C), 30.9 (d, 2C); IR (KBr) νmax/cm−1 3395, 3306, 2921, 2857, 1708, 1647, 1512, 1384, 1369, 1317, 1249, 721, 701. HRMS (nano UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C49H51N5O9 854.3765; Found 854.3761. [α]D20 +13.4 (c 0.810, 10% CH3OH/CH2Cl2). PhthAd-Gly-Phe-Phe[3,4-(OMe)2]-OH (5). Prepared from 22 (703 mg, 1.47 mmol) and 11 (647 mg, 1.38 mmol) according to the above general procedure in 48% yield (purified on a C-18 reverse phase MPLC by use of 30−100% H2O/CH3OH as eluent); 468 mg (48%); colorless solid; mp 222−225 °C; 1H NMR (300 MHz, CD3OD) δ/ ppm 7.77−7.74 (m, 4H), 7.26−7.11 (m, 5H), 6.90−6.74 (m, 3H), 4.52 (dd, J = 9.3, 4.7 Hz, 1H), 4.46 (dd, J = 7.8, 4.9 Hz, 1H), 3.92 (d, J = 16.5 Hz, 1H), 3.82 (s, 3H), 3.71 (s, 3H), 3.74 (d, J = 16.5 Hz, 1H), 3.22−3.02 (m, 2H), 3.00−2.80 (m, 2H), 2.61 (d, J = 12.5 Hz, 2H), 2.55 (s, 2H), 2.45 (d, J = 12.5 Hz, 2H), 2.26 (s, 2H), 1.87 (d, J = 12.4 Hz, 2H), 1.75−1.84 (m, 3H), 1.70 (d, J = 12.6 Hz, 1H); 13C NMR (75 MHz, CD3OD) δ/ppm 180.5 (s, 1C), 173.0 (s, 1C), 172.0 (s, 1C), 170.9 (s, 1C), 167.4 (s, 2C), 150.1 (s, 1C), 149.1 (s, 1C), 138.2 (s, 1C), 135.3 (d, 2C), 133.0 (s, 1C), 132.2 (s, 2C), 130.1 (d, 2C), 129.6 (d, 2C), 127.8 (d, 1C), 123.5 (d, 2C), 122.9 (d, 1C), 114.6 (d, 1C), 113.0 (d, 1C), 61.3 (s, 1C), 56.6 (d/q, 2C), 56.5 (d/q, 2C), 44.1 (s, 1C), 43.8 (t, 1C), 42.0 (t, 1C), 40.2 (t, 2C), 38.7 (t, 2C), 38.4 (t, 1C), 38.2 (t, 1C), 36.1 (t, 1C), 30.9 (d, 2C); IR (KBr) νmax/cm−1 3393, 2920, 1705, 1644, 1519, 1467, 1313, 1046, 822, 721, 642. HRMS (nano-UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C41H44N4O9 737.3187; Found 737.3189. [α]D20 −23.1 (c 0.39, 10% CH3OH/CH2Cl2). Boc-Phe-Phe(OMe)-OH (8). Prepared from 7 (147 mg, 0.75 mmol) according to the above general procedure in quantitative yield (purified on a column of silica gel with 0−10% CH3OH/CH2Cl2 as eluent); 333 mg (100%); colorless solid; mp 149−151 °C; 1H NMR (300 MHz, DMSO-d6) δ/ppm 12.65 (s, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.30−7.12 (m, 7H), 6.88−6.79 (m, 3H), 4.45−4.35 (m, 1H), 4.22−4.11 (m, 1H), 3.71 (s, 3H), 3.05−2.80 (m, 3H), 2.71−2.58 (m, 1H), 1.26 (s, 9H); 13C NMR (75 MHz, DMSO) δ/ppm 172.9 (s, 1C), 171.8 (s, 1C), 158.1 (s, 1C), 155.2 (s, 1C), 138.2 (s, 1C), 130.4 (d, 2C), 129.3 (d, 2C), 128.3 (s, 1C), 128.1 (d, 2C), 126.3 (d, 1C), 113.7 (d, 2C), 78.2 (s, 1C), 55.8 (d, 1C), 55.1 (q, 1C), 53.7 (d, 1C), 37.5 (t, 1C), 36.1 (t, 1C), 28.2 (q, 3C); IR (KBr) νmax/cm−1 3338, 3295, 2979, 2936, 1681, 1648, 1516, 1256, 1162, 1020, 699. Boc-Phe-Phe[3,4-(OMe)2]-OH (9). Prepared from 7 (170 mg, 0.75 mmol) according to the above general procedure in 70% yield (purified on a column of silica gel with 0−10% CH3OH/CH2Cl2 as eluent); 250 mg, 70%; colorless solid; mp 186−188 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.29−7.13 (m, 5H), 6.88−6.72 (m, 3H), 4.72−4.59 (m, 1H), 4.32−4.22 (m, 1H), 3.81 (s, 3H), 3.78 (s, 3H), 3.18−2.92 (m, 3H), 2.78−2.65 (m, 1H), 1.33 (br. s, 9H); 13C NMR (150 MHz, CD3OD) δ/ppm 174.3 (s, 1C), 150.4 (s, 1C), 149.5 (s, 1C), 138.6 (s, 1C), 130.9 (s, 1C), 130.3 (d, 2C), 129.4 (d, 2C), 127.6 (d, 1C), 122.9 (d, 1C), 114.3 (d, 1C), 113.1 (d, 1C), 80.6 (s, 1C), 57.4 (d, 1C), 56.5 (q, 2C), 55.0 (d, 1C), 39.2 (t, 1C), 38.1 (t, 1C), 28.6 (q, 3C); two singlets were not observed; IR (KBr) νmax/cm−1 3408, 3336, 2926, 2852, 1687, 1652, 1516, 1270, 1164, 1136. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C25H32N2O7 495.2107; Found 495.2110. Boc-Phe-Phe-Phe-OH (15).25 Prepared from 14 (539 mg, 1.27 mmol) according to the above general procedure in 75% yield (purified on a column of silica gel with 0−10% CH3OH/CH2Cl2 as eluent); 530 mg, 75%; colorless solid; mp 168−169 °C; 1H NMR (300 MHz, CDCl3) δ/ppm 7.30−7.15 (m, 11H), 7.14−7.02 (m, 5H), 6.86 (br. s, 1H), 6.58 (d, J = 6.4 Hz, 1H), 4.95 (br. s, 1H), 4.69−4.58 (m, 2H), 4.37 (br. s, 2H), 3.15 (dd, J = 5.4, 14.0 Hz, 1H), 3.03−2.82 (m, 5H), 1.35 (br. s, 9H); 13C NMR (75 MHz, CDCl3) δ/ppm 173.3 (s, 1C), 171.7 (s, 1C), 170.7 (s, 1C), 155.8 (s, 1C), 136.4 (s, 1C),

(d, 1C), 123.6 (d, 2C), 61.3 (t, 1C), 55.9 (d, 1C), 55.7 (d, 1C), 55.2 (d, 1C), 44.0 (s, 1C), 43.9 (s, 1C), 42.2 (t, 1C), 40.3 (t, 2C), 38.9 (t, 2C), 38.7 (t, 1C), 38.5 (t, 1C), 38.4 (t, 1C), 36.2 (t, 1C), 31.0 (d, 2C); IR (KBr) νmax/cm−1 3391, 3307, 2915, 1857, 1771, 1668, 1520, 1367, 1313, 1078, 875, 721, 698, 642. HRMS (nano UPLC-ESIqTOF) m/z: [M + H]+ Calcd for C48H49N5O8 824.3659; Found 824.3657. [α]D20 −18.2 (c 0.770, 10% CH3OH/CH2Cl2). General Procedure for the Activation of Carboxylic Acid by DCC-NHS.25 A flask was filled with a Boc-protected amino acid (10 mmol), DCC (11 mmol), NHS (11 mmol), and dry CH2Cl2 (25 mL). The reaction mixture was stirred from −5 to 0 °C for 5 h and left in a refrigerator overnight. The next day the precipitate was filtered off, and the filtrate was evaporated. The residue was dissolved in ethyl acetate, and the precipitate was filtered off. The filtrate was washed with 0.5 M NaHCO3, H2O, 0.5 M HCl, and H2O. After drying over anhydrous MgSO4 and filtration, the solvent was removed on a rotary evaporator. The crude succinimido-activated amino acids/ peptides 7,23,25 14,23,25 and 2223 were used in the coupling step without additional purification. General Procedure for the Peptide Coupling via Sucinimide Activation.25 A flask was charged with amino acid (2.5 mmol), NaHCO3 (5.0 mmol), and THF−H2O (1:1, 20 mL). To the mixture a solution of succinimide-activated amino acid in THF (2.6 mmol in 15 mL) was added dropwise, and the reaction mixture was stirred at rt 2 days. THF was removed on a rotary evaporator, and the reaction mixture was acidified with 0.5 M HCl to pH ≈ 2. The product was extracted with ethyl acetate (3 × 30 mL). The organic extracts were washed with H2O and dried over anhydrous Na2SO4. After filtration and evaporation of the solvent, the residue was chromatographed on a column of silica gel with 0−10% CH3OH/CH2Cl2 as eluent or on a C-18 reverse phase MPLC by use of 30−100% H2O/CH3OH as eluent for peptides 3 and 5. PhthAd-Gly-Phe-Phe-OH (1). Prepared from 22 (541 mg, 1.13 mmol) and 13 (440 mg, 1.03 mmol) according to the above general procedure in 87% yield (purified on a C-18 reverse phase MPLC by use of 30−100% H2O/CH3OH as eluent). PhthAd-Gly-Phe-Phe-Phe-OH (2). Prepared from 22 (724 mg, 1.51 mmol) and 18 (786 mg, 1.37 mmol) according to the above general procedure in 55% yield (purified on a C-18 reverse phase MPLC by use of 30−100% H2O/CH3OH as eluent). PhthAd-Gly-Phe-Phe(OMe)-OH (3). Prepared from 22 (696 mg, 1.45 mmol) and 10 (607 mg, 1.32 mmol) according to the above general procedure in 58% yield (purified on a C-18 reverse phase MPLC by use of 30−100% H2O/CH3OH as eluent); 545 mg (58%); colorless solid; mp 208−210 °C; 1H NMR (300 MHz, CD3OD) δ/ ppm 7.75 (s, 4H), 7.29−7.05 (m, 7H), 6.79 (d, J = 8.7 Hz, 2H), 4.68−4.54 (m, 2H), 3.86−3.65 (m, 5H), 3.17−3.06 (m, 2H), 3.01− 2.81 (m, 2H), 2.65−2.54 (m, 4H), 2.46 (d, J = 11.9 Hz, 2H), 2.27 (s, 2H), 1.95−1.75 (m, 5H), 1.70 (d, J = 12.6 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ/ppm 170.9 (s, 1C), 160.0 (s, 2C), 138.1 (s, 1C), 135.2 (d, 2C), 133.1 (s, 2C), 131.4 (d, 2C), 130.4 (d, 2C), 130.3 (s, 1C), 129.5 (d, 2C), 127.8 (d, 1C), 123.5 (d, 2C), 114.8 (d, 2C), 61.4 (s, 1C), 55.7 (q, 1C, d, 1C), 55.5 (d, 1C), 44.1 (s/t, 1C), 43.8 (s/t, 1C), 42.2 (t, 1C), 40.3 (t, 2C), 38.9 (t, 2C), 38.7 (t, 1C), 37.7 (t, 1C), 36.2 (t, 1C), 31.0 (d, 2C), three singlets were not observed; IR (KBr) νmax/cm−1 3399, 2917, 1705, 1646, 1516, 1435, 1383, 1344, 1040, 725 cm−1. HRMS (nano UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C40H42N4O8 707.3081; Found 707.3090. [α]D20 +3.61 (c 0.553, 10% CH3OH/CH2Cl2). PhthAd-Gly-Phe-Phe-Phe(OMe)-OH (4). Prepared from 22 (701 mg, 1.46 mmol) and 19 (803 mg, 1.33 mmol) according to the above general procedure in 32% yield (purified on a C-18 reverse phase MPLC by use of 30−100% H2O/CH3OH as eluent); 363 mg (32%); colorless solid; mp 147−150 °C; 1H NMR (300 MHz, CD3OD) δ/ ppm 7.76−7.67 (m, 4H), 7.26−7.02 (m, 12H), 6.77 (d, J = 8.5 Hz, 2H), 4.64−4.56 (m, 1H), 4.56−4.46 (m, 2H), 3.80 (d, J = 16.3 Hz, 1H), 3.74−3.64 (m, 4H), 3.18−3.08 (m, 2H), 2.99−2.88 (m, 3H), 2.85−2.73 (m, 1H), 2.62−2.51 (m, 4H), 2.43 (d, J = 12.0 Hz, 2H), 2.24 (br.s, 2H), 1.90−1.72 (m, 5H), 1.67 (d, J = 12.6 Hz, 1H); 13C NMR (75 MHz, CD3OD) δ/ppm 180.3 (s, 1C), 176.3 (s, 1C), 173.4 14914


Article


Hz, 1H), 5.10 (d, J = 12.4 Hz, 1H), 5.00 (d, J = 8.0 Hz, 1H), 4.65− 4.55 (m, 1H), 3.84 (s, 3H), 3.77 (s, 3H), 3.03 (d, J = 5.5 Hz, 2H), 1.41 (m, 9H); 13C NMR (150 MHz, CD3OD) δ/ppm 173.6 (s, 1C), 157.8 (s, 1C), 150.4 (s, 1C), 149.4 (s, 1C), 137.1 (s, 1C), 131.0 (s, 1C), 129.5 (d, 2C), 129.24 (d, 1C/2C), 129.20 (d, 1C/2C), 122.7 (d, 1C), 114.2 (d, 1C), 113.1 (d, 1C), 80.6 (s, 1C), 67.8 (t, 1C), 56.8 (d, 1C), 56.5 (q, 1C), 56.4 (q, 1C), 38.3 (t, 1C), 28.7 (q, 3C); IR (KBr) νmax/cm−1 3379, 2977, 2938, 1746, 1715, 1518, 1368, 1264, 1238, 1163, 1027, 752, 699. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C23H29NO6 438.1893; Found 438.1897. General Procedure for the Boc Deprotection.25 Bocprotected amino acid or peptide (10 mmol) was dissolved in dry CH2Cl2 (60 mL) under inert N2 atmosphere and cooled to 0 °C. TFA (115 mL, 1.5 mol) was added dropvise during 1 h, and the stirring was continued 1 h at 0 °C and 2 h at rt. After the reaction was completed, the solvent and TFA were removed by evaporation. To the residue, cold diethyl ether or hexane was added (∼40 mL) whereupon the product precipitated. The product was filtered off via a sinter funnel and dried in a desiccator over P2O5 and KOH over 2 days. TFA×H-Phe-Phe-OH (13).25 Prepared according to the general procedure for the Boc deprotection from 12 (1.03 g, 2.27 mmol) in 95% yield; 921 mg (95%); colorless solid; mp 78 °C; 1H NMR (300 MHz, DMSO-d6) δ/ppm 8.86 (d, 1H, J = 7.8 Hz), 8.15 (br.s, 3H), 7.36−7.20 (m, 10H), 4.57−4.48 (m, 1H), 4.05−3.99 (m, 1H), 3.16− 3.07 (m, 2H), 3.00−2.86 (m, 2H); 13C NMR (150 MHz, DMSO-d6) δ/ppm 172.2 (s, 1C), 168.2 (s, 1C), 137.1 (s, 1C), 134.7 (s, 1C), 129.5 (d, 2C), 129.1 (d, 2C), 128.5 (d, 2C), 128.3 (d, 2C), 127.2 (d, 1C), 126.6 (d, 1C), 53.8 (d, 1C), 53.2 (d, 1C), 36.9 (t, 1C), 36.7 (t, 1C). TFA×H-Phe-Phe-Phe-OH (18). Prepared according to the general procedure for the Boc deprotection from 15 (530 mg, 0.95 mmol) in 97% yield; 526 mg (97%); colorless solid; mp 125−126 °C; 1H NMR (300 MHz, DMSO-d6) δ/ppm 8.66 (d, J = 8.2 Hz, 1H), 8.51 (d, J = 7.8 Hz, 1H), 8.09 (br. s, 3H), 7.36−7.08 (m, 15H), 4.69−4.59 (m, 1H), 4.55−4.45 (m, 1H), 3.99−3.91 (m, 1H), 3.14−2.75 (m, 6H); 13 C NMR (150 MHz, DMSO-d6) δ/ppm 172.5 (s, 1C), 170.5 (s, 2C), 137.4 (s, 1C), 137.3 (s, 2C), 129.5 (d, 2C), 129.2 (d, 2C), 129.0 (d, 2C), 128.4 (d, 2C), 128.1 (d, 2C), 128.0 (d, 2C), 127.0 (d, 1C), 126.34 (d, 1C), 126.31 (d, 1C), 53.8 (d, 1C), 53.4 (d, 1C), 53.2 (d, 1C), 37.6 (t, 1C), 36.7 (t, 2C); IR (KBr) νmax/cm−1 3290, 3010, 2918, 1637, 1529, 1453, 1200, 1132, 747, 699. TFA×H-Phe-Phe(OMe)-OH (10). Prepared according to the general procedure for the Boc deprotection from 8 (333 mg, 0.75 mmol) in 89% yield; 305 mg (89%); colorless solid; mp 127−130 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.37−7.27 (m, 5H), 7.16 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 4.66 (dd, J = 5.2, 8.6 Hz, 1H), 4.08 (dd, J = 5.2, 8.7 Hz, 1H), 3.75 (s, 3H), 3.30−3.26 (m, 1H), 3.18 (dd, J = 5.2, 14.1 Hz, 1H), 3.03−2.93 (m, 2H); 13C NMR (150 MHz, CD3OD) δ/ppm 174.1 (s, 1H), 169.6 (s, 1C), 160.2 (s, 1C), 135.5 (s, 1C), 131.2 (d, 2C), 130.6 (d, 2C), 130.1 (d, 2C), 130.1 (s, 1C), 128.9 (d, 1C), 115.0 (d, 2C), 55.7 (q+(d/q), 2C), 55.5 (d/q, 1C), 38.5 (t, 1C), 37.5 (t, 1C); IR (KBr) νmax/cm−1 3317, 2914, 2361, 1715, 1699, 1653, 1636, 1558, 1540, 1514, 1457, 1249, 1203, 1178, 1139, 1095, 1035, 838. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C21H21F3N2O5 461.1300; Found 461.1280. TFA×H-Phe-Phe-Phe(OMe)-OH (19). Prepared according to the general procedure for the Boc deprotection from 16 (1.33 g, 2.25 mmol) in 93% yield; 1.26 g (93%); colorless solid; mp 187−190 °C; 1 H NMR (300 MHz, CD3OD) δ/ppm 7.30−7.12 (m, 12H), 6.81 (d, J = 8.6 Hz, 2H), 4.70 (dd, J = 5.5, 8.8 Hz, 1H), 4.61 (dd, J = 4.9, 8.4 Hz, 1H), 3.99 (dd, J = 4.9, 8.9 Hz, 1H), 3.66 (s, 3H), 3.20−3.09 (m, 3H), 2.97−2.82 (m, 3H); 13C NMR (150 MHz, CD3OD) δ/ppm 174.3 (s, 1C), 172.8 (s, 1C), 169.4 (s, 1C), 160.0 (s, 1C), 138.1 (s, 1C), 135.4 (s, 1C), 131.4 (d, 2C), 130.5 (d, 2C), 130.3 (d, 2C), 130.2 (s, 1C), 130.16 (d, 2C), 129.5 (d, 2C), 128.9 (d, 1C), 127.9 (d, 1C), 114.9 (d, 2C), 56.0 (d, 1C), 55.5 (d, 1C), 55.41 (d/q, 1C), 55.39 (d/ q, 1C), 39.0 (t, 1C), 38.6 (t, 1C), 37.6 (t, 1C); IR (KBr) νmax/cm−1 3293, 3032, 2926, 1695, 1673, 1644, 1514, 1201, 1137, 747, 723, 701.

136.2 (s, 1C), 136.0 (s, 1C), 129.5 (d, 2C), 129.4 (d, 4C), 128.9 (d, 2C), 128.85 (d, 2C), 128.8 (d, 2C), 127,3 (d, 1C), 127.23 (d, 1C), 127.20 (d, 1C), 54.5 (d, 1C), 54.1 (d, 2C), 38.2 (t, 1C), 37.4 (t, 1C), 29.9 (t, 1C), 28.3 (q, 3C), one signal (s, 1C) is covered by the CDCl3 signal. Boc-Phe-Phe-Phe(OMe)-OH (16). Prepared from 14 (371 mg, 1.90 mmol) according to the above general procedure in 76% yield (purified on a column of silica gel with 0−10% CH3OH/CH2Cl2 as eluent); 851 mg (76%); colorless solid; mp = 142−145 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.30−7.06 (m, 12H), 6.80 (d, J = 8.6 Hz, 2H), 4.67−4.52 (m, 2H), 4.27−4.17 (m, 1H), 3.69 (s, 3H), 3.11 (dd, J = 5.4, 13.9 Hz, 2H), 2.98−2.83 (m, 3H), 2.71−2.60 (m, 1H), 1.33 (br. s, 9H); 13C NMR (150 MHz, CD3OD) δ/ppm 174.2 (s, 1C), 174.0 (s, 1C), 172.9 (s, 1C), 160.1 (s, 1C), 157.5 (s, 1C), 138.7 (s, 1C), 138.1 (s, 1C), 131.4 (d, 2C), 130.5 (d, 2C), 130.3 (d, 2C), 130.0 (s, 1C), 129.40 (d, 2C), 129.36 (d, 2C), 127.7 (d, 1C), 127.6 (d, 1C), 114.9 (d, 2C), 80.6 (s, 1C), 57.3 (d 1C), 55.6 (d, 1C), 55.5 (d, 1C), 55.3 (q, 1C), 39.2 (t, 1C), 39.0 (t, 1C), 37.6 (t, 1C), 28.6 (q, 3C); IR (KBr) νmax/cm−1 3406, 3313, 2931, 1648, 1510, 1369, 1250, 1176, 699. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C33H39N3O7 612.2686; Found 612.2667. Boc-Phe-Phe-Phe[3,4-(OMe)2]-OH (17). Prepared from 14 (1.20 g, 2.36 mmol) according to the above general procedure in 89% yield (purified on a column of silica gel with 0−10% CH3OH/CH2Cl2 as eluent); 1.18 g (89%); colorless solid; mp 155−157 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.28−7.12 (m, 10H), 6.86−6.72 (m, 3H), 4.68−4.58 (m, 2H), 4.24−4.18 (m, 1H), 3.80 (s, 3H), 3.73 (s, 3H), 3.16−3.06 (m, 2H), 3.00−2.84 (m, 3H), 2.68−2.60 (m, 1H), 1.33 (s, 9H); 13C NMR (150 MHz, CD3OD) δ/ppm 174.3 (s, 1C), 173.9 (s, 1C), 172.9 (s, 1C), 157.5 (s, 1C), 150.2 (s, 1C), 149.3 (s, 1C), 138.7 (s, 1C), 138.1 (s, 1C), 130.9 (s, 1C), 130.5 (d, 2C), 130.3 (d, 2C), 129.4 (d, 2C), 129.3 (d, 2C), 127.7 (d, 1C), 127.6 (d, 1C), 122.8 (d, 1C), 114.1 (d, 1C), 112.9 (d, 1C), 80.6 (s, 1C), 57.3 (d, 1C), 56.4 (q, 1C), 56.3 (q, 1C), 55.5 (d, 1C), 55.3 (d, 1C), 39.15 (t, 1C), 39.10 (t, 1C), 38.0 (t, 1C), 28.6 (q, 3C); IR (KBr) νmax/cm−1 3332, 3030, 2971, 2928, 2476, 1721, 1684, 1642, 1517, 1420, 1267, 1158, 1029, 700 cm−1. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C34H41N3O8 642.2791; Found 642.2800. L-Boc-Phe[3,4-(OMe)2]-OH (23). A flask (100 mL) was charged with K2CO3 (1.840 g, 13.32 mmol) and dioxane−H2O (1:1, 30 mL). The mixture was cooled to 0 °C, and amino acid H-Phe[3,4(OMe)2]-OH (1.00 g, 4.44 mmol) was added, followed by a solution of Boc2O (0.969 g, 4.44 mmol) in dioxane (4 mL). The stirring was continued at rt over 24 h. When the reaction was completed, H2O (35 mL) was added as well as saturated KHSO4 until pH 4 was reached. An extraction with ethyl acetate (3 × 50 mL) was carried out, and the extracts were dried over anhydrous MgSO4. After filtration and evaporation of the solvent, the crude product was obtained quantitatively and used without further purification. Yellow oil; 1H NMR (300 MHz, CDCl3) δ/ppm 9.33 (br. s, 1H), 6.81−6.70 (m, 3H), 5.05 (d, J = 7.58 Hz, 1H), 4.57 (d, J = 11.9, 5.3 Hz, 1H), 3.85 (s, 3H), 3.15−2.95 (m, 2H), 1.44−1.38 (m, 9H); 13C NMR (150 MHz, CD3OD) δ/ppm 175.4 (s, 1C), 157.7 (s, 1C), 150.3 (s, 1C), 149.4 (s, 1C), 131.5 (s, 1C), 122.8 (d, 1C), 114.3 (d, 1C), 113.1 (d, 1C), 80.5 (s, 1C), 56.5 (d, 1C), 56.4 (q, 1C), 56.3 (q, 1C), 38.3 (t, 1C), 28.7 (q, 3C); IR (KBr) νmax/cm−1 3397, 2983, 2941, 1753, 1720, 1543, 1372, 1264, 1184, 1027, 745, 707. L-Boc-Phe[3,4-(OMe)2]-OBn (24). A flask (100 mL) was charged with Boc-protected amino acid 23 (1.78 g, 4.44 mmol, 75% purity), NaHCO3 (373 mg, 4.44 mmol), DMF−dioxane (1:1, 20 mL), and benzyl bromide (580 μL, 4.88 mmol). The reaction mixture was stirred at 90 °C over 24 h. When the reaction was completed, the solvent was removed on a rotary evaporator and 1 M HCl (30 mL) was added. An extraction with ethyl acetate (3 × 25 mL) was conducted, and the extracts were washed with 25 mL of 2 M HCl, H2O, sat. NaHCO3, and H2O. The extracts were dried over anhydrous MgSO4 and filtered, and the solvent was evaporated to afford the product (1.64 g, 89%) in the form of a yellow oil. Yellow oil; mp 182 °C, 1H NMR (300 MHz, CDCl3) δ/ppm 7.40−7.25 (m, 5H), 6.72 (d, J = 7.6 Hz, 1H), 6.63−6.56 (m, 2H), 5.18 (d, J = 12.4 14915


Article

The Journal of Organic Chemistry HRMS (MALDI-TOF-TOF) m/z: [M + Na] + Calcd for C30H30F3N3O6 608.1984; Found 608.1990. TFA×H-Phe-Phe[3,4-(OMe)2]-OH (11). Prepared according to the general procedure for the Boc deprotection from 9 (250 mg, 0.53 mmol) in 95% yield; 244 mg (95%); colorless solid; mp 202−204 °C; 1 H NMR (600 MHz, CD3OD) δ/ppm 7.38−7.28 (m, 5H), 6.87− 6.85 (m, 2H), 6.79 (dd, J = 1.7, 8.1 Hz, 1H), 4.70 (dd, J = 5.3, 8.3 Hz, 1H), 4.07 (dd, J = 5.1, 8.6 Hz, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.28 (dd, J = 5.1, 14.5 Hz, 1H), 3.18 (dd, J = 5.1, 14.2 Hz, 1H), 3.04−2.94 (m, 2H); 13C NMR (150 MHz, CD3OD) δ/ppm 169.6 (s, 1C), 150.5 (s, 1C), 149.6 (s, 1C), 135.4 (s, 1C), 131.0 (s, 1C), 130.6 (d, 2C), 130.1 (d, 2C), 128.9 (d, 1C), 122.8 (d, 1C), 114.1 (d, 1C), 113.1 (d, 1C), 56.5 (q, 2C), 55.6 (d, 1C), 55.5 (d, 1C), 38.5 (t, 1C), 38.0 (t, 1C), one singlet was not observed; IR (KBr) νmax/cm−1 3421, 1654, 1584, 1519, 1402, 1333, 1261, 1232, 1196, 1135, 1024, 703. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C22H23F3N2O6 491.1406; Found 491.1420. TFA×H-Phe-Phe-L-Phe[3,4-(OMe)2]-OBn (20). Prepared according to the general procedure for the Boc deprotection from 17 (785 mg, 1.27 mmol) in 90% yield; 810 mg (90%); colorless solid; mp 186− 189 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.27−7.02 (m, 2H), 6.78 (s, 1H), 6.70 (s, 2H), 4.57 (br. s, 2H), 3.86 (br. s, 1H), 3.71 (s, 3H), 3.59 (s, 3H), 3.14−2.96 (m, 3H), 2.91−2.81 (m, 1H), 2.81− 2.70 (m, 2H); 13C NMR (150 MHz, CD3OD) δ/ppm 198.0 (s, 1C), 150.2 (s, 1C), 149.3 (s, 1C), 138.2 (s, 1C), 131.5 (s, 1C), 130.5 (d, 2C), 130.3 (d, 2C), 130.1 (d, 2C), 129.5 (d, 2C), 128.8 (d, 1C), 128.8 (d, 1C), 123.0 (d, 1C), 114.6 (d, 1C), 113.0 (d, 1C), 56.6 (q, 1C), 56.4 (q, 1C), 55.4 (d, 3C), 38.2 (t, 1C), 38.8 (t, 1C), 38.5 (t, 1C), two singlets are not observed; IR (KBr) νmax/cm−1 3065, 1667, 1638, 1515, 1259, 1199, 1134, 1028, 742, 695. L-TFA×H-Phe[3,4-(OMe)2]-OBn (25). Prepared according to the general procedure for the Boc deprotection from 24 (1.63 g, 3.95 mmol) in 80% yield; 1.36 g (80%); yellow solid; mp 130−132 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.38−7.28 (m, 5H), 6.85 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 2.0 Hz, 1H), 6.71 (dd, J = 2.0, 8.2 Hz, 1H), 5.25 (d, J = 12.1 Hz, 1H), 5.22 (d, J = 12.1 Hz, 1H), 4.33 (t, J = 6.8 Hz, 1H) 3.80 (s, 3H), 3.75 (s, 3H), 3.25−3.06 (m, 2H); 13C NMR (75 MHz, CD3OD) δ/ppm 170.1 (s, 1C), 150.9 (s, 1C), 150.3 (s, 1C), 136.2 (s, 1C), 129.8 (d, 2C), 129.7 (d, 2C), 129.7 (d, 1C), 127.6 (s, 1C), 123.0 (d, 1C), 114.0 (d, 1C), 113.3 (d, 1C), 69.1 (t, 1C), 56.44 (q, 1C), 56.41 (q, 1C), 37.1 (t, 1C); IR (KBr) νmax/cm−1 3197, 2943, 2835, 1748, 1676, 1608, 1518, 1453, 1420, 1404, 1262, 1203, 1131, 1022, 833, 809, 800, 768, 752, 739, 721, 697. HRMS (MALDITOF-TOF) m/z: [M + H]+ Calcd for C20H20F3NO5 412.1372; Found 412.1389. TFA×H-Phe-Phe-OBn (27). Prepared according to the general procedure for the Boc deprotection from 26 (1.60 g, 3.18 mmol) in 72% yield; 1.19 g (72%); colorless solid, mp 154 °C; 1H NMR (300 MHz, DMSO-d6) δ/ppm 9.02 (d, J = 7.5 Hz, 1H), 8.09 (s, 3H), 7.42−7.15 (m, 15H), 5.12 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.4 Hz, 1H), 4.70−4.60 (m, 1H), 4.07−3.97 (m, 1H), 3.15−2.95 (m, 3H), 2.90−2.80 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ/ppm 170.6 (s, 1C), 168.4 (s, 1C), 136.5 (s, 1C), 135.5 (s, 1C), 134.6 (s, 1C), 129.5 (d, 2C), 129.1 (d, 2C), 128.6 (d, 2C), 128.4 (d, 4C), 128.2 (d, 1C), 128.1 (d, 2C), 127.2 (d, 1C), 126.8 (d, 1C), 66.4 (t, 1C), 53.9 (d, 1C), 53.1 (d,1C), 36.9 (t, 1C), 36.7 (t, 1C); IR (KBr) νmax/cm−1 3297, 3067, 3031, 2934, 1674, 1517, 1454, 1202, 1137, 839, 747, 722, 700 cm−1. TFA×H-Phe-Phe-Phe-OBn (29).41 Prepared according to the general procedure for the Boc deprotection from 28 (458 mg, 0.69 mmol) in 72% yield; 339 mg (72%); colorless solid, mp 167 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.36−7.14 (m, 20H), 5.10 (s, 2H), 4.73−4.64 (m, 2H), 4.04−3.95 (m, 1H), 3.21−2.78 (m, 6H); 13 C NMR (75 MHz, CD3OD) δ/ppm 172.9 (s, 1C), 171.4 (s, 1C), 169.4 (s, 1C), 138.0 (s, 1C), 137.9 (s, 1C), 137.0 (s, 1C), 135.4 (s, 1C), 130.5 (d, 2C), 130.4 (d, 2C), 130.3 (d, 2C), 120.2 (d, 2C), 129.60 (d, 2C), 129.57 (d, 2C), 129.55 (d, 2C), 129.5 (d, 2C), 129.4 (d, 1C), 128.9 (d, 1C), 127.9 (d, 1C), 127.87 (d, 1C), 60.1 (t, 1C), 55.9 (d, 1C), 55.5 (d, 1C), 55.4 (d, 1C), 39.1 (t, 1C), 38.6 (t, 1C), 38.5 (t, 1C).

TFA×H-Phe-Phe-Phe[3,4-(OMe)2]-OBn (31). Prepared according to the general procedure for the Boc deprotection from 30 (360 mg, 0.51 mmol) in 93% yield; 343 mg (93%); colorless solid; mp 181− 184 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.35−7.17 (m, 15H), 6.82 (d, J = 1.6 Hz, 1H), 6.79 (d, J = 8.2 Hz, 1H), 6.73 (dd, J = 1.6, 8.2 Hz, 1H), 5.11 (s, 2H), 4.73−4.68 (m, 2H), 4.01−3.99 (m, 1H), 3.76 (s, 3H), 3.71 (s, 3H), 3.16−3.05 (m, 3H), 2.97−2.83 (m, 3H); 13 C NMR (150 MHz, CD3OD) δ/ppm 172.8 (s, 1C), 172.5 (s, 1C), 169.4 (s, 1C), 150.4 (s, 1C), 149.5 (s, 1C), 138.0 (s, 1C), 137.0 (s, 1C), 135.4 (s, 1C), 130.7 (s, 1C), 130.5 (d, 2C), 130.3 (d, 2C), 130.1 (d, 2C), 129.5 (d, 2C), 129.4 (d, 2C), 128.9 (d, 2C/1C), 127.9 (d, 1C/2C), 122.8 (d, 1C), 114.3 (d, 1C), 113.1 (d, 1C), 68.1 (t, 1C), 56.5 (q, 1C), 56.4 (q, 1C), 55.9 (d, 1C), 55.6 (d, 1C), 55.4 (d, 1C), 39.1 (t, 1C), 38.6 (t, 1C), 38.2 (t, 1C); IR (KBr) νmax/cm−1 3361, 3264, 3063, 3032, 2957, 2937, 1671, 1516, 1264, 1239, 1203, 1134, 1030, 744, 721, 698. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C38H38F3N3O7 728.2560; Found 728.2550. General Procedure for the Peptide Coupling via HBTUHOBT Activation.26 A flask under N2 atmosphere was charged with N-Boc-protected amino acid (1 mmol) triethylamine (TEA, 2.1 mmol), HBTU (1.1 mmol), HOBt (1.1 mmol), and dry DMF or CH2Cl2 (5 mL). The reaction mixture was stirred at rt 10 min. TFA salt of Bn-protected amino acid (or amine) (1.1 mmol) was added, and the stirring was continued 3 days. When the reaction was completed, brine (50 mL) was added, and the suspension was extracted with diethyl ether (1 × 50 mL). The organic layer was washed with 1 M HCl (50 mL), H2O (50 mL), saturated NaHCO3 (50 mL), and again with H2O (50 mL). After drying over anhydrous MgSO4, the solution was filtered and the solvent was removed on a rotary evaporator to afford the crude product that was purified by column chromatography on silica gel with 0−10% CH3OH/CH2Cl2 as eluent. Boc-Phe-Phe-OBn (26).42 Prepared from Boc-Phe-OH (273 mg, 1.03 mmol) and TFA×H-Phe-OBn (400 mg, 1.08 mmol) according to the above general procedure in 88% yield; 458 mg (88%); colorless solid; 1H NMR (300 MHz, CDCl3) δ/ppm 7.42−7.11 (m, 13H), 6.93−6.86 (m, 2H), 6.26 (d, J = 7.3 Hz, 1H), 5.10 (s, 2H), 5.00−4.87 (br. s, 1H), 4.86−4.75 (m, 1H), 4.38−4.24 (m, 1H), 3.13−2.94 (m, 4H), 1.39 (s, 9H); 13C NMR (75 MHz, CDCl3) δ/ppm 170.92 (s, 1C), 170.88 (s, 1C), 136.6 (s, 1C), 135.6 (s, 1C), 135.2 (s, 1C), 129.5 (d, 2C), 129.4 (d, 2C), 128.8 (d, 2C), 128.76 (d, 2C), 128.71 (d, 2C), 128.67 (d, 2C), 127.2 (d, 1C), 127.13 (d, 1C), 127.08 (d, 1C), 67.4 (t, 1C), 53.5 (d, 2C), 38.5 (t, 1C), 38.1 (t, 1C), 28.4 (q, 3C), two singlets were not seen. Boc-Phe-Phe-Phe-OBn (28).41 Prepared from Boc-Phe-Phe-OH (12, 244 mg, 0.59 mmol) and TFA×H-Phe-OBn (240 mg, 0.65 mmol) according to the above general procedure in 77% yield; 475 mg (77%); colorless solid; 1H NMR (300 MHz, CDCl3) δ/ppm 7.39−7.11 (m, 11H), 7.07−7.01 (m, 2H), 6.97−6.90 (m, 2H), 6.44 (d, J = 7.5 Hz, 1H), 6.27−5.15 (m, 1H), 5.08 (s, 2H), 4.87−4.78 (m, 1H), 4.78−4.69 (m, 1H), 4.58−4.49 (m, 1H), 4.34−4.23 (m, 1H), 3.08−2.83 (m, 6H), 1.37 (s, 9H). Boc-Phe-Phe-Phe[3,4-(OMe)2]-OBn (30). Prepared from Boc-PhePhe-OH (12, 500 mg, 1.21 mmol) and TFA×H-Phe[3,4-(OMe)2]OBn (25, 400 mg, 1.27 mmol) according to the above general procedure in 87% yield; 1.05 g (87%); colorless solid; mp 175−177 °C; 1H NMR (300 MHz, CDCl3) δ/ppm 7.40−7.11 (m, 14H), 7.10− 7.02 (m, 2H), 6.67 (d, J = 8.2 Hz, 1H), 6.54 (d, J = 1.7 Hz, 1H), 6.49−6.39 (m, 2H), 6.14 (d, J = 6.2 Hz, 1H), 5.09 (s, 2H), 4.72 (dd, J = 6.3, 13.6 Hz, 1H), 4.53 (dd, J = 7.2, 13.7 Hz, 1H), 4.27 (dd, J = 6.4, 13.3 Hz, 1H), 3.82 (s, 3H), 3.74 (s, 3H), 3.05−2.84 (m, 6H), 1.37 (s, 9H); 13C NMR (150 MHz, CDCl3) δ/ppm 171.1 (s, 1C), 170.9 (s, 1C), 170.1 (s, 1C), 149.1 (s, 1C), 148.3 (s, 1C), 136.5 (s, 1C), 136.3 (s, 1C), 135.3 (s, 1C), 129.43 (d, 2C), 129.40 (d, 2C), 128.9 (d, 2C), 128.8 (d, 2C), 128.7 (d, 1C), 128.6 (d, 1C), 128.5 (d, 1C), 128.1 (s, 1C), 127.22 (d, 2C), 127.21 (d, 2C), 121.5 (d, 1C), 112.4 (d, 1C), 111.3 (d, 1C), 67.2 (t, 1C), 55.98 (q, 1C), 55.97 (q, 1C), 54.5 (d, 1C), 53.8 (d, 2C), 38.2 (t, 1C), 38.0 (t, 1C), 37.7 (t, 1C), 28.4 (q, 3C), two singlets were not seen; IR (KBr) νmax/cm−1 3292, 3063, 3029, 2973, 2930, 1745, 1690, 1646, 1516, 1266, 1165, 1025, 741, 14916


Article

The Journal of Organic Chemistry 700. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C41H47N3O8 732.3261; Found 732.3242. PhthAd-Gly-Phe-Phe-OBn (1-OBn). Prepared from 21 (250 mg, 0.65 mmol) and TFA×H-Phe-Phe-OBn (27, 354 mg, 0.686 mmol) according to the above general procedure. The pure product (289 mg, 62%) was obtained after column chromatography on silica gel using 2.5−7% CH3OH/CH2Cl2 as eluent. Colorless solid; mp 89−92 °C; 1 H NMR (300 MHz, CDCl3) δ/ppm 7.80−7.60 (m, 4H), 7.40−7.10 (m, 12H), 6.95 (br. s, 2H), 6.80 (d, J = 6.6 Hz, 1H), 6.57−6.44 (m, 2H), 5.29 (s, 1H), 5.08 (s, 2H), 4.85−4.75 (m, 1H), 4.70−4.58 (m, 1H), 3.80 (d, J = 3.4 Hz, 2H), 3.13−2.91 (m, 4H), 2.61−2.35 (m, 6H), 2.29 (br. s, 2H), 1.94−1.60 (m, 6H); 13C NMR (75 MHz, CDCl3) δ/ppm 177.3 (s, 1C), 170.9 (s, 1C), 170.3 (s, 1C), 169.7 (s, 1C), 169.2 (s, 2C), 136.4 (s, 1C/2C), 135.8 (s, 1C/2C), 135.2 (s, 1C), 133.9 (d, 2C), 131.9 (s, 1C), 129.4 (d, 4C), 128.8 (d, 2C), 128.7 (d, 2C), 128.6 (d, 4C), 127.1 (d, 2C), 122.8 (d, 2C), 67.3 (t, 1C), 60.3 (t, 1C), 54.4 (d, 1C), 53.5 (d, 1C), 47.7 (s, 1C), 43.3 (t, 1C), 42.9 (s, 1C), 41.2 (t, 2C), 39.3 (t, 2C), 38.0 (t, 1C), 37.9 (t, 1C), 35.2 (t, 1C), 29.5 (d, 2C); IR (KBr) νmax/cm−1 3393, 3288, 3063, 3030, 2919, 2851, 1739, 1706, 1656, 1522, 1437, 1371, 1313, 1242, 1078, 740, 722, 701. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C46H46N4O7 789.3264; Found 789.3292. PhthAd-Gly-Phe-Phe-Phe-OBn (2-OBn). Prepared from 21 (87 mg, 0.23 mmol) and TFA×H-Phe-Phe-Phe-OBn (29, 200 mg, 0.24 mmol) according to the above general procedure. The pure product (42 mg, 20%) was obtained after column chromatography on silica gel using 2.5−7% CH3OH/CH2Cl2 as eluent and preparative TLC with CH3OH/EtOEt/CH2Cl2 (2:28:70) as eluent. Colorless solid; mp 102−104 °C; 1H NMR (300 MHz, CDCl3) δ/ppm 7.94 (d, J = 7.7 Hz, 1H), 7.74−7.67 (m, 2H), 7.67−7.59 (m, 2H), 7.54 (d, J = 7.7 Hz, 1H), 7.30−7.00 (m, 20H), 6.93 (d, J = 8.4 Hz, 1H), 6.93 (t, J = 4.0 Hz, 1H), 5.04 (br. s, 2H), 5.02−4.94 (m, 1H), 4.88−4.80 (m, 1H), 4.76−4.66 (m, 1H), 3.95−3.74 (m, 2H), 3.18−2.92 (m, 4H), 2.88− 2.77 (m, 4H), 2.64−2.52 (m, 4H), 2.44 (d, J = 13.0 Hz, 2H), 2.29 (br.s, 2H), 1.91 (d, J = 12.1 Hz, 2H), 1.85−1.73 (m, 3H), 1.65 (d, J = 12.0 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ/ppm 177.0 (s, 1C), 171.0 (s, 1C), 170.8 (s, 1C), 170.3 (s, 1C), 169.6 (s, 2C), 168.7 (s, 1C), 136.6 (s, 1C), 136.4 (s, 1C), 136.3 (s, 1C), 135.4 (s, 1C), 133.8 (d, 2C), 131.9 (s, 2C), 129.48 (d, 1C), 129.46 (d, 2C), 129.41 (d, 2C), 129.38 (d, 2C), 129.3 (d, 2C), 128.6 (d, 2C), 128.5 (d, 2C),128.34 (d, 2C), 128.30 (d, 2C), 126.98 (d, 1C), 126.96 (d, 1C), 126.8 (d, 1C), 122.7 (d, 2C), 67.0 (t, 1C), 60.3 (t, 1C), 54.3 (d, 1C), 54.1 (d, 1C), 53.8 (d, 1C), 43.3 (s, 1C), 42.8 (t/s, 1C), 41.2 (s/t, 1C), 39.34 (t, 1C), 39.32 (t, 1C), 39.1 (t, 1C), 39.0 (t, 1C), 38.1(t, 1C), 38.0 (t, 1C), 37.95 (t, 1C), 35.2 (t, 1C), 29.6 (d, 2C); IR (KBr) νmax/cm−1 3393, 3288, 3063, 3030, 2919, 2851, 1739, 1706, 1656, 1522, 1437, 1371, 1313, 1242, 1078, 740, 722, 701. HRMS (MALDITOF-TOF) m/z: [M + Na]+ Calcd for C55H55N5O8 936.3948; Found 936.3939. PhthAd-Gly-Phe-Phe-Phe[3,4-(OMe)2]-OBn (32). Prepared from 21 (500 mg, 1.21 mmol) and TFA×H-Phe-Phe-Phe[3,4-(OMe)2]OBn (31, 200 mg, 0.28 mmol) according to the above general procedure. The pure product 32 (228 mg, 85%) was obtained after column chromatography on silica gel by use of 30% EtOAc/CH2Cl2 as eluent and increasing polarity to 5% CH3OH/CH2Cl2. Colorless solid; mp 118−120 °C; 1H NMR (300 MHz, CDCl3) δ/ppm 7.89 (d, J = 6.4 Hz, 1H), 7.72−7.68 (m, 2H), 7.64−7.61 (m, 2H), 7.53 (d, J = 6.8 Hz, 1H), 7.40−6.97 (m, 15H), 6.81 (d, J = 8.0 Hz, 1H), 6.74 (br. s, 1H), 6.68 (s, 1H), 6.63 (s, 2H), 5.28 (s, 1H), 5.10−4.98 (m, 3H), 4.86−4.76 (m, 1H), 4.74−4.65 (m, 1H), 4.00−3.80 (m, 2H), 3.75 (s, 3H), 3.73 (s, 3H), 3.08−2.99 (m, 3H), 2.89−2.78 (m. 3H), 2.64 (s, 2H), 2.62−2.53 (m, 2H), 2.50−2.41 (m, 2H), 2.29 (br. s, 2H), 1.97− 1.88 (m, 2H), 1.86−1.73 (m, 3H), 1.65 (d, J = 12.5 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ/ppm 177.1 (s, 1C), 171.2 (s, 1C), 170.6 (s, 1C), 170.2 (s, 1C), 169.6 (s, 1C/2C), 168.6 (s, 1C/2C), 148.9 (s, 1C), 148.0 (s, 1C), 136.5 (s, 1C), 136.2 (s, 2C), 135.4 (s, 1C), 133.8 (d, 2C), 131.9 (s, 1C), 129.39 (d, 2C), 129.38 (d, 2C), 128.8 (s, 1C), 128.6 (d, 2C), 128.5 (d, 2C), 128.3 (d, 3C), 128.1 (d, 2C), 127.0 (d, 1C), 126.8 (d, 1C), 122.7 (d, 2C), 121.4 (d, 1C), 112.5 (d, 2C), 111.1 (d, 1C), 66.8 (t, 1C), 60.4 (s, 1C), 60.2 (t, 1C), 55.82 (q, 1C),

55.79 (q, 1C), 54.4 (d, 1C), 54.1 (d, 2C), 43.3 (s, 1C), 42.8 (t, 1C), 41.2 (t, 1C), 39.3 (t, 2C), 39.2 (t, 1C), 39.0 (t, 1C), 38.0 (t, 2C), 37.9 (t, 1C), 35.2 (t, 1C), 29.5 (d, 2C); IR (KBr) νmax/cm−1 3412, 3297, 2913, 1853, 1707, 1637, 1515, 1315, 1265, 721, 698. PhthAd-Gly-Phe-Phe-Phe[3,4-(OMe)2]-OH (6). A flask (20 mL) was charged with Bn-ester 32 (100 mg, 0.10 mmol), 10% Pd/C (25 mg), and dry methanol (5 mL). The reaction mixture was purged with N2 for 15 min. Et3SiH (0.165 mL, 1.0 mmol) was added dropwise, and the stirring was continued over 1 h. The progress of the reaction was monitored on TLC by use of 5% CH3OH/CH2Cl2 as eluent. Upon completion, Pd/C was filtered off through filter paper (blue ribbon), and the solvent was removed on a rotary evaporator to afford the pure product 6 (84 mg, quantitatively) in the form of a colorless solid. Colorless solid; mp 177−180 °C; 1H NMR (300 MHz, DMSO-d6) δ/ppm 8.17 (d, J = 7.6 Hz, 1H), 7.85−7.74 (m, 5H), 7.65 (t, J = 7.4 Hz, 1H), 7.51 (s, 1H), 7.21−7.08 (m, 13H), 4.50−4.37 (m, 3H), 3.87 (s, 3H), 3.75 (s, 3H), 3.70 (d, J = 16.4 Hz, 1H), 3.51 (d, J = 16.4 Hz, 1H), 3.08−2.58 (m, 6H), 2.50−2.43 (m, 4H), 2.35 (d, J = 12.8 Hz, 2H), 2.20 (s, 2H), 1.82−1.55 (m, 6H); 1H NMR (300 MHz, CD3OD) δ/ppm 7.82 (s, 1H), 7.78−7.65 (m, 4H), 7.64 (s, 1H), 7.36 (s, 2H), 7.26−7.05 (m, 8H), 7.04−6.96 (m, 2H), 6.82 (s, 1H), 6.76 (s, 2H), 4.58−4.73 (m, 3H), 3.83 (s, 3H), 3.78 (s, 3H), 3.77−3.69 (m, 2H), 3.22−3.05 (m, 2H), 2.97−2.77 (m, 4H), 2.57−2.41 (m, 6H), 2.27 (s, 2H), 1.83−1.58 (m, 6H); 13C NMR (150 MHz, DMSO-d6) δ/ppm 175.7 (s, 1C), 170.2 (s, 1C), 169.4 (s, 1C), 168.5 (s, 2C), 168.3 (s, 1C), 151.8 (s, 1C), 146.1 (s, 1C), 140.8 (s, 1C), 137.4 (s, 1C), 137.2 (s, 1C), 133.8 (d, 2C), 130.7 (s, 2C), 128.7 (d, 2C), 128.6 (d, 2C), 127.5 (d, 2C), 127.4 (d, 2C), 125.5 (d, 2C), 121.9 (d, 2C), 113.7 (d, 1C/2C), 107.3 (d, 1C/2C), 59.3 (s, 1C), 55.5 (q, 1C), 55.2 (q, 1C), 53.7 (d, 1C), 53.2 (d, 1C), 53.1 (d, 1C), 41.6 (t, 1C), 40.3 (s, 1C), 38.3 (t, 2C), 37.0 (t, 1C), 36.9 (t, 2C), 35.0 (t, 1C), 34.3 (t, 1C), 32.6 (t, 1C), 28.5 (d, 2C), one singlet was not observed; IR (KBr) νmax/cm−1 3400, 2915, 1707, 1646, 1516, 1382, 1317, 1265, 724, 700. HRMS (nano-UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C50H53N5O10 884.3871; Found 884.3864. [α]D20 +26.50 (c 0.113, 10% CH3OH/CH2Cl2). Irradiation ExperimentsGeneral. In a quartz Erlenmeyer flask, peptide (0.1 mmol) and K2CO3 (0.05 mmol) were dissolved in acetone−H2O (3:1). The solution was purged with N2 for 20 min, sealed, and irradiated 10−30 min in a reactor at 300 nm (8 lamps, 1 lamp 8 W). During the irradiation, the sample was continuously stirred by a magnet bar and cooled by a fan. The solvent was removed on a rotary evaporator, and the residue was chromatographed on a column of silica gel. Irradiation of 1. Peptide 1 (53 mg, 0.08 mmol) dissolved in 75 mL acetone−H2O (3:1) was irradiated for 10 min. The crude photoreaction mixture was purified by chromatography on a column of silica gel using 5% CH3OH/CH2Cl2 and HOAc/CH3OH/CH2Cl2 (1:9:90) as eluent. Pure product 33 was isolated after preparative thin layer chromatography on silica gel with 5% CH3OH/CH2Cl2 as eluent. Traces of 39 were identified in the crude photolysis mixture by HPLC based on a comparison of the retention time with the authentic sample obtained by an alternative synthetic method (see Schemes 7 and 8). (6S,9S)-1(1,3)-Adamantana-2,7,10,13-tetraaza-4(1,2)-benzena6,9-dibenzyl-3,5,8,11,14-tetraoxocyclotetradecaphane (33). Colorless solid (8 mg, 15%); mp 173−175 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.42−7.31 (m, 2H), 7.26 (dt, J = 1.1, 7.4 Hz, 1H), 7.16−7.03 (m, 6H), 7.02−6.96 (m, 3H), 6.83−6.78 (m, 2H), 5.13− 5.05 (m, 1H), 4.64−4.57 (m, 1H), 4.16 (d, J = 15.1 Hz, 1H), 3.33 (d, J = 15.1 Hz, 1H), 2.91−2.76 (m, 2H), 2.73−2.62 (m, 2H), 2.43 (d, J = 12.1 Hz, 1H), 2.23−2.08 (m, 3H), 2.05 (d, J = 8.9 Hz, 1H), 1.95− 1.70 (m, 4H), 1.70−1.50 (m, 5H); 1H NMR (600 MHz, CDCl3) δ/ ppm 7.32−7.20 (m, 10H), 7.04−7.00 (m, 4H), 6.88−6.85 (m, 2H), 5.78 (s, 1H), 5.13−5.09 (m, 1H), 4.82−4.76 (m, 1H), 4.61−4.55 (m, 1H), 3.31 (d, J = 14.0 Hz, 1H), 3.07 (dd, J = 6.4, 13.3 Hz, 1H), 2.95 (dd, J = 8.5, 13.3 Hz, 1H), 2.86 (dd, J = 5.2, 14.1 Hz, 1H), 2.80 (d, J = 12.3 Hz, 1H), 2.55−2.47 (m, 2H), 2.36−2.30 (m, 2H), 2.05 (br. s, 1H), 1.91 (d, J = 12.2 Hz, 2H), 1.79 (d, J = 11.4 Hz, 2H), 1.69 (d, J = 10.5 Hz, 1H), 1.64−1.54 (m, 3H), 1.50 (d, J = 12.0 Hz, 1H); 13C 14917


Article

The Journal of Organic Chemistry NMR (150 MHz, CDCl3) δ/ppm 178.0 (s, 1C), 170.5 (s, 1C), 167.2 (s, 1C), 139.91 (s, 1C), 139.89 (s, 1C), 136.8 (s, 1C), 136.3 (s, 1C), 130.8 (d, 1C), 130.6 (d, 1C), 129.7 (d, 2C), 129.3 (d, 2C), 128.7 (d, 3C), 128.2 (d, 2C), 127.0 (d, 1C), 126.7 (d, 1C), 125.5 (d, 1C), 60.0 (d, 1C), 54.9 (d, 1C), 53.4 (s, 1C), 44.4 (t/s, 1C), 43.1 (t/s, 1C), 42.6 (t, 1C), 42.4 (t, 1C), 40.2 (t, 1C), 39.9 (t, 1C), 39.2 (t, 1C), 36.4 (t, 1C), 36.2 (t, 1C), 35.5 (t, 1C), 29.5 (d, 1C), 29.4 (d, 1C), one singlet at ∼200 ppm was not observed; IR (KBr) νmax/cm−13407, 3323, 2925, 2856, 1652, 1524, 1455, 1382. HRMS (nano-UPLC-ESIqTOF) m/z: [M + H]+ Calcd for C38H40N4O5 633.3077; Found 633.3085. [α]D20 +8.1 (c 0.266, 10% CH3OH/CH2Cl2). Irradiation of 2. Peptide 2 (50 mg, 0.06 mmol) in 75 mL of acetone−H2O (3:1) was irradiated 20 min. Pure photoproduct 34 was isolated after column chromatography on silica gel by use of Et2O/ CH3OH/CH2Cl2 (20:5:75) as eluent, followed by preparative thin layer chromatography on silica gel with CH3OH/EtOAc/hexane/ CH2Cl2 (2:4:4:90) as eluent. Traces of 40 were identified in the crude photolysis mixture by HPLC based on a comparison of the retention time with the authentic sample obtained by an alternative synthetic method (see Schemes 7 and 8). (6R,9S,12S)-1(1,3)-Adamantana-2,7,10,13,14-pentaaza-4(1,2)benzena-6,9,12-tribenzyl-3,5,8,11,14,17-pentaoxocycloheptadecaphane (34). Colorless solid (8 mg, 16%), mp 166−170 °C; 1H NMR (600 MHz, CD3CN) δ/ppm 7.69−7.66 (m, 1H), 7.57−7.51 (m, 2H), 7.50−7.48 (m, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.27−7.15 (m, 9H), 7.15−7.12 (m, 2H), 7.08−7.04 (m, 4H), 6.97 (d, J = 6.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.65 (t, J = 4.4 Hz, 1H), 6.48 (s, 1H), 5.27 (ddd, J = 4.4, 8.2, 8.9 Hz, 1H), 4.38 (ddd, J = 5.1, 8.4, 8.7 Hz, 1H), 4.31 (ddd, J = 5.5, 5.6, 8.9 Hz, 1H), 3.87 (dd, J = 4.8, 16.5 Hz, 1H), 3.67 (dd, J = 4.8, 16.5 Hz, 1H), 3.15 (dd, J = 4.4, 14.2 Hz, 1H), 2.94 (dd, J = 4.9, 14.1 Hz, 1H), 2.92−2.86 (m, 2H), 2.81−2.70 (m, 2H), 2.60 (d, J = 11.6 Hz, 1H), 2.44 (d, J = 11.6 Hz, 1H), 2.21−2.15 (m, 2H), 1.98 (d, J = 11.8 Hz, 1H), 1.92−1.85 (m, 3H), 1.84 (s, 2H), 1.76 (s, 2H), 1.70 (d, J = 12.6 Hz, 1H), 1.66 (d, J = 12.6 Hz, 1H); 13C NMR (150 MHz, CD3CN) δ/ppm 201.9 (s, 1C), 177.9 (s, 1C), 171.9 (s, 1C), 171.48 (s, 1C), 171.46 (s, 1C), 169.5 (s, 1C), 139.2 (s, 1C), 138.7 (s, 1C), 138.5 (s, 1C), 138.1 (s, 1C), 137.8 (s, 1C), 132.4 (d, 1C), 130.6 (d, 1C), 130.5 (d, 2C), 130.3 (d, 2C), 130.1 (d, 2C), 129.42 (d, 2C), 129.40 (d, 2C), 129.3 (d, 2C), 129.0 (d, 1C), 128.3 (d, 1C), 127.7 (d, 1C), 127.6 (d, 1C), 127.5 (d, 1C), 59.6 (d, 1C), 57.2 (d, 1C), 55.5 (d, 1C), 53.9 (s, 1C), 43.5 (s/t, 1C), 43.3 (s/t, 1C), 43.2 (t, 1C), 42.2 (t, 1C), 40.7 (t, 1C), 38.7 (t, 1C), 38.6 (t, 1C), 38.2 (t, 1C), 38.1 (t, 1C), 37.6 (t, 1C), 36.1 (t, 1C), 30.33 (d, 1C), 30.30 (d, 1C); IR (KBr) νmax/cm−1 3401, 2919, 2860, 1652, 1522, 745, 705, 612. HRMS (nanoUPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C47H49N5O6 780.3761; Found 780.3763. [α]D20 +15.3 (c 0.350, 10% CH3OH/CH2Cl2). Irradiation of 3. Peptide 3 (30 mg, 0.04 mmol) and K2CO3 (2.7 mg, 0.02 mmol) in 45 mL of acetone−H2O (3:1) were irradiated 15 min. Pure photoproduct 35 was isolated after chromatography on a thin layer of silica gel with 5% CH3OH/CH2Cl2 as eluent. (6S,9S)-1(1,3)-Adamantana-2,7,10,13-tetraaza-4(1,2)-benzena6-(4-methoxyphenylmethyl)-9-benzyl-3,5,8,11,14-tetraoxocyclotetradecaphane (35). Colorless crystals (12 mg, 40%); mp 169−171 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.50 (dd, J = 0.5, 7.4 Hz, 1H), 7.45 (ddd, J = 1.0, 7.5, 7.6 Hz, 1H), 7.37 (ddd, J = 1.0, 7.5, 7.6 Hz, 1H), 7.24−7.11 (m, 6H), 6.80 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 8.6 Hz, 2H), 5.14 (t, J = 6.8 Hz, 1H), 4.69 (dd, J = 6.0, 8.1 Hz, 1H), 4.25 (d, J = 15.0 Hz, 1H), 3.70 (s, 3H), 3.42 (d, J = 15.0 Hz, 1H), 3.00−2.91 (m, 2H), 2.77 (d, J = 12.3 Hz, 1H), 2.69 (d, J = 6.8 Hz, 2H), 2.52 (d, J = 12.3 Hz, 1H), 2.30−2.25 (m, 1H), 2.23−2.14 (m, 3H), 2.01−1.93 (m, 2H), 1.92−1.84 (m, 2H), 1.76−1.62 (m, 4H); 1 H NMR (600 MHz, acetone-d6) δ/ppm 7.66−7.59 (m, 2H), 7.57− 7.53 (m, 1H), 7.46 (s, 1H), 7.43−7.38 (m, 2H), 7.28−7.19 (m, 5H), 7.17−7.14 (m, 1H), 7.01 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H), 6.62 (d, J = 8.4 Hz, 2H), 5.21−5.17 (m, 1H), 4.85−4.79 (m, 1H), 4.40−4.34 (m, 1H), 3.81−3.74 (m, 1H), 3.71 (s, 3H), 3.22 (dd, J = 4.0, 14.5 Hz, 1H), 2.70 (dd, J = 6.9, 14.4 Hz, 1H), 2.64 (dd, J = 5.6, 14.3 Hz, 1H), 2.51 (d, J = 11.7 Hz, 1H), 2.28−2.24 (m, 2H), 2.19 (s, 1H), 2.12−2.08 (m, 2H), 1.93−1.90 (m, 3H), 1.73−1.59 (m, 6H);

C NMR (75 MHz, CD3OD) δ/ppm 202.9 (s, 1C), 180.4 (s, 1C), 172.3 (s, 1C), 171.7 (s, 1C), 170.4 (s, 1C), 160.0 (s, 1C), 140.0 (s, 1C), 138.1 (s, 1C), 137.9 (s, 1C), 131.8 (d, 1C), 131.3 (d, 1C), 131.2 (d, 2C), 130.7 (d, 2C), 129.4 (d, 3C), 129.3 (s, 1C), 128.2 (d, 1C), 127.8 (d, 1C), 114.8 (d, 2C), 60.8 (d, 1C), 55.7 (d/q, 1C), 55.6 (d/q, 1C), 54.3 (s, 1C), 44.8 (s/t, 1C), 44.7 (s/t, 1C), 43.8 (t, 1C), 42.7 (t, 1C), 40.8 (t, 1C), 40.2 (t, 1C), 38.8 (t, 1C), 37.6 (t, 1C), 37.3 (t, 1C), 36.5 (t, 1C), 30.8 (d, 2C); 13C NMR (150 MHz, acetone-d6) δ/ ppm 202.4 (s, 1C), 178.4 (s, 1C), 171.0 (s, 1C), 170.6 (s, 1C), 168.0 (s, 1C), 159.3 (s, 1C), 140.9 (s, 1C), 138.3 (s, 1C), 138.0 (s, 1C), 131.2 (d, 2C), 130.9 (d, 1C), 130.85 (d, 1C), 130.6 (d, 2C), 129.1 (s, 1C), 129.02 (d, 2C), 128.99 (d, 1C), 127.5 (d, 1C), 127.3 (d, 1C), 114.3 (d, 2C), 60.7 (d, 1C), 55.4 (d/q, 1C), 55.1 (d/q, 1C), 53.8 (s, 1C), 44.7 (t, 1C), 44.0 (t, 1C), 43.3 (s, 1C), 42.6 (t, 1C), 40.6 (t, 1C), 40.5 (t, 1C), 38.3 (t, 1C), 37.5 (t, 1C), 37.1 (t, 1C), 36.3 (t, 1C), 30.4 (d, 1C), 30.3 (d, 1C); IR (KBr) νmax/cm−13413, 3322, 2927, 2856, 1667, 1644, 1522, 1250, 1250, 1032, 736. HRMS (nanoUPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C39H42N4O6 663.3183; Found 663.3191. [α]D20 −20.5 (c 0.400, 10% CH3OH/ CH2Cl2). Irradiation of 4. Peptide 4 (100 mg, 0.12 mmol) and K2CO3 (8.0 mg, 0.06 mmol) in 100 mL of acetone−H2O (3:1) were irradiated for 20 min. The pure photoproduct was isolated after column chromatography through 1 cm of silica with 0−10% CH3OH/ CH2Cl2 as eluent. Fractions containing products were further purified by semipreparative HPLC (37% H2O/CH3OH, 0.1% TFA, over 5 min, 37−0% H2O/CH3OH, 0.1% TFA, over 30 min, CH3OH, over 5 min, flow 3 mL/min) and then by preparative thin layer chromatography with CH3OH/EtOAc/CH2Cl2 (3:10:87) as eluent to afford one diastereomer of cyclic peptide 36 (9 mg, 9%) and a mixture of peptide diastereomers 36 (10 mg, 10%). (6R,9S,12S)-1(1,3)-Adamantana-2,7,10,13,14-pentaaza-4(1,2)benzena-6-(4-methoxyphenylmethyl)-9,12-dibenzyl-3,5,8,11,14,17pentaoxocycloheptadecaphane (36). Colorless solid (9 mg, 9%); mp 189 °C; 1H NMR (600 MHz, CDCl3) δ/ppm 7.69 (d, J = 7.2 Hz, 1H), 7.50−7.43 (m, 2H), 7.40 (d, J = 7.1 Hz, 1H), 7.31−7.16 (m, 7H), 7.10 (d, J = 7.2 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 7.2 Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 6.41 (s, 1H), 6.27 (br. s, 1H), 5.49 (s, 1H), 5.41−5.35 (m, 1H), 4.62−4.57 (m, 1H), 4.46−4.40 (m, 1H), 3.81 (d, J = 16.5 Hz, 1H), 3.75 (s, 3H), 3.72 (d, J = 16.5 Hz, 1H), 3.13 (dd, J = 6.6, 13.9 Hz, 1H), 3.07−3.01 (m, 2H), 2.99 (dd, J = 7.2, 14.3 Hz, 1H), 2.93−2.87 (m, 2H), 2.77 (d, J = 11.9 Hz, 1H), 2.72 (d, J = 11.9 Hz, 1H), 2.30 (s, 1H), 2.24 (s, 1H), 1.99 (d, J = 12.7 Hz, 1H), 1.96 (d, J = 11.8 Hz, 1H), 1.89−1.78 (m, 6H), 1.70 (s, 2H); 13C NMR (75 MHz, CD3OD) δ/ppm 202.3 (s, 1C), 179.6 (s, 1C), 173.1 (s, 1C), 172.8 (s, 1C), 172.4 (s, 1C), 171.9 (s, 1C), 160.0 (s, 1C), 139.8 (s, 1C), 138.2 (s, 1C), 138.0 (s, 1C), 137.0 (s, 1C), 133.2 (d, 1C), 131.4 (d, 2C), 130.7 (d, 1C), 130.4 (s, 1C), 130.2 (d, 2C), 130.1 (d, 2C), 129.6 (d, 2C), 129.5 (d, 2C), 129.3 (d, 1C), 128.5 (d, 1C), 127.9 (d, 1C), 127.8 (d, 1C), 115.0 (d, 2C), 59.7 (d, 1C), 57.4 (d, 1C), 56.0 (d/q, 1C), 55.7 (d/q, 1C), 54.2 (s, 1C), 43.7 (t, 1C), 43.5 (s, 1C), 43.2 (t, 1C), 42.1 (t, 1C), 40.8 (t, 1C), 38.85 (t, 1C), 38.77 (t, 1C), 38.2 (t, 1C), 37.6 (t, 1C), 37.5 (t, 1C), 36.5 (t, 1C), 30.7 (d, 2C); IR (KBr) νmax/cm−13400, 3308, 3061, 3034, 2916, 2857, 1656, 1516, 1446, 1253, 1172, 1032, 736. HRMS (nanoUPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C48H51N5O7 810.3867; Found 810.3870. [α]D20 +12.5 (c 0.310, 10% CH3OH/ CH2Cl2) (pure isolated diastereomer). Irradiation of 5. Peptide 5 (200 mg, 0.27 mmol) and K2CO3 (19 mg, 0.14 mmol) in 100 mL of acetone−H2O (3:1) were irradiated for 20 min. Pure photoproducts 37 and 43 were isolated after column chromatography on silica gel with CH 3 OH/EtOAc/CH 2 Cl 2 (10:20:60) as eluent, followed by preparative TLC on silica gel and EtOEt/CH3OH/CH2Cl2 (20:4:76). PhthAd-Gly-Phe-[Et-Ph3,4-(OMe)2] (43). Colorless crystals, 4 mg, 2%; mp 188−191 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.78− 7.66 (m, 4H), 7.27−7.11 (m, 5H), 6.83−6.78 (m, 2H), 6.70 (dd, J = 1.8, 8.2 Hz, 1H), 4.54 (dd, J = 5.8, 8.1 Hz, 1H), 3.80 (s, 3H), 3.77 (s, 3H), 3.75−3.70 (m, 2H), 3.09 (dd, J = 5.8, 13.7 Hz, 1H), 2.89 (dd, J = 8.2, 13.9 Hz, 1H), 2.67 (t, J = 7.3 Hz, 2H), 2.63−2.39 (m, 6H), 13

14918


Article

The Journal of Organic Chemistry 2.27 (br. s, 2H), 1.94−1.76 (m, 6H), 1.70 (d, J = 12.6 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ/ppm 180.2 (s, 1C), 173.1 (s, 1C), 171.6 (s, 1C/2C), 170.9 (s, 1C/2C), 150.4 (s, 1C), 149.0 (s, 1C), 138.2 (s, 2C), 135.2 (d, 2C), 133.4 (s, 1C), 133.1 (s, 1C), 130.3 (d, 2C), 129.6 (d, 2C), 127.9 (d, 1C), 123.5 (d, 2C), 122.2 (d, 1C), 113.8 (d, 1C) 113.1 (d, 1C), 61.3 (s, 1C), 56.5 (q, 1C), 56.4 (q, 1C), 55.9 (d, 1C), 44.1 (t, 1C), 42.3 (t, 2C), 40.4 (t, 1C), 40.3 (t, 1C), 38.9 (t, 1C), 38.8 (t, 1C), 38.7 (t, 1C), 36.3 (t, 1C), 36.0 (t, 1C), 31.0 (d, 2C), one signlet was not observed; IR (KBr) νmax/cm−13434, 2957, 2924, 2854, 2707, 1684, 1676, 1654, 1647, 1638, 1561, 1457, 1264, 1207, 1137. HRMS (nanoUPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C40H44N4O7 693.3288; Found 693.3290. [α]D20 +5.1 (c 0.120, 10% CH3OH/CH2Cl2). (6S,9S)-1(1,3)-Adamantana-2,7,10,13-tetraaza-4(1,2)-benzena6-(3,4-dimethoxyphenylmethyl)-9-benzyl-3,5,8,11,14-tetraoxocyclotetradecaphane (37). Colorless crystals, 12 mg, 6%; mp 177−180 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.53−7.32 (m, 3H), 7.23−7.06 (m, 6H), 6.68 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 1.9 Hz, 1H), 6.51 (dd, J = 1.9, 8.1 Hz, 1H), 5.15 (dd, J = 7.0 Hz, 1H), 4.67 (dd, J = 6.5, 7.5 Hz, 1H), 4.21 (d, J = 15.0 Hz, 1H), 3.74 (s, 1H), 3.71 (s, 3H), 3.46 (d, J = 15.0 Hz, 1H), 2.97−2.92 (m, 1H), 2.78−2.67 (m, 3H), 2.54−2.46 (m, 1H), 2.31−2.12 (m, 4H), 2.03−1.83 (m, 4H), 1.78−1.60 (m, 5H); 13C NMR (150 MHz, CD3OD) δ/ppm 203.0 (s, 1C), 180.4 (s, 1C), 172.3 (s, 1C), 171.6 (s, 1C), 170.4 (s, 1C), 150.2 (s, 1C), 149.4 (s, 1C), 140.1 (s, 1C), 138.0 (s, 1C), 137.8 (s, 1C), 131.8 (d, 1C), 131.2 (d, 1C), 130.6 (d, 2C), 130.3 (s, 1C), 129.4 (d, 1C), 129.3 (d, 2C), 128.1 (d, 1C), 127.7 (d, 1C), 122.8 (d, 1C), 114.1 (d, 1C), 112.9 (d, 1C), 60.8 (d, 1C), 56.44 (q, 1C), 56.42 (q, 1C), 55.8 (d, 1C), 54.4 (s, 1C), 44.8 (t, 1C), 44.7 (t, 1C), 43.8 (s, 1C), 42.7 (t, 1C), 40.8 (t, 1C), 40.1 (t, 1C), 39.5 (t, 1C), 37.6 (t, 1C), 37.3 (t, 1C), 36.5 (t, 1C), 30.8 (d, 1C), 30.7 (d, 1C); IR (KBr) νmax/cm−1 3426, 2921, 1852, 1655, 1647, 1637, 1540, 1528, 1385, 1091. HRMS (nanoUPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C40H44N4O7 693.3288; Found 693.3285. [α]D20 −19.1 (c 0.350, 10% CH3OH/CH2Cl2). Irradiation of 6. Peptide 6 (49 mg, 0.06 mmol) and K2CO3 (3.8 mg, 0.03 mmol) in 75 mL of acetone−H2O (3:1) were irradiated for 20 min. The pure photoproducts were isolated after column chromatography on silica gel with 3% CH3OH/CH2Cl2 followed by semipreparative HPLC (53% H2O/CH3OH, 0.1% TFA, over 5 min, 53−0% H2O/CH3OH, 0.1% TFA, over 20 min, CH3OH, over 5 min, flow 4 mL/min) and TLC on silica gel using 4% CH3OH/CH2Cl2 as eluent. (6R,9S,12S)-1(1,3)-Adamantana-2,7,10,13,14-pentaaza-4(1,2)benzena-6-(3,4-dimethoxyphenylmethyl)-9,12-dibenzyl3,5,8,11,14,17-pentaoxocycloheptadecaphane (38). Colorless solid; 6 mg (12%); mp 196−197 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.87−7.81 (m, 1H), 7.68−7.52 (m, 3H), 7.30−7.03 (m, 10H), 6.84− 6.77 (m, 2H), 6.76−6.68 (m, 1H), 5.42 (dd, J = 4.2, 9.9 Hz, 1H), 4.49 (dd, J = 5.2, 9.5 Hz, 1H), 4.32 (dd, J = 4.6, 8.8 Hz, 1H), 3.93 (d, J = 16.7 Hz, 1H), 3.81−3.67 (m, 7H), 3.24−2.55 (m, 6H), 2.50−2.42 (m, 1H), 2.30−2.20 (m, 2H), 2.09−1.68 (m, 11H); 13C NMR (75 MHz, acetone-d6) δ/ppm 191.5 (s, 1C), 174.2 (s, 1C), 171.3 (s, 2C), 139.6 (s, 1C), 138.7 (s, 1C), 138.3 (s, 1C), 131.9 (d, 1C), 131.5 (s, 1C), 130.1 (d, 2C), 130.0 (d, 1C), 129.95 (d, 2C), 129.2 (d, 2C), 129.1 (d, 2C), 128.7 (d, 1C), 128.1 (d, 1C), 127.4 (d, 1C), 127.3 (d, 1C), 122.3 (d, 1C), 114.3 (d, 1C), 112.8 (d, 1C), 59.9 (d, 1C), 57.2 (d, 1C), 56.11 (q, 1C), 56.09 (q, 1C), 55.2 (d, 1C), 53.6 (s, 1C), 43.4 (t/s, 1C), 43.1 (s/t, 1C), 43.0 (t, 1C), 42.1 (t, 1C), 40.6 (t, 1C), 40.0 (t, 1C), 38.7 (t, 1C), 38.6 (t, 1C), 38.0 (t, 1C), 37.4 (t, 1C), 36.4 (t, 1C), one doublet (covered by the solvent) and five singlets were not observed; IR (KBr) νmax/cm−1 3427, 2927, 2854, 1654, 1647, 1637, 1541, 1517, 1457, 1265, 1089. HRMS (nanoUPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C49H53N5O8 840.3972; Found 840.3971. [α]D20 −13.1 (c 0.210, 10% CH3OH/CH2Cl2). Preparation of Decarboxylation Products 39 and 40. BocPhe-(Et-Ph) (45). Prepared from Boc-Phe-OH (300 mg, 1.13 mmol) and 2-phenylethylamine (96 μL, 1.24 mmol) according to the above general procedure for the peptide coupling via HBTU-HOBT activation. The pure product (251 mg, 60%) was obtained after

column chromatography on silica gel by use of 10% EtOEt/CH2Cl2 as eluent. Colorless solid; mp 141−142 °C; 1H NMR (600 MHz, CDCl3) δ/ppm 7.35−7.17 (m, 8H), 7.03 (d, J = 6.9 Hz, 2H), 5.68 (br. s, 1H), 5.00 (br. s, 1H), 4.26−4.20 (m, 1H), 3.50−3.43 (m, 1H), 3.42−3.35 (m, 1H), 3.10−2.97 (m, 2H), 2.73−2.66 (m, 1H), 2.65− 2.55 (m, 1H), 1.40 (s, 9H); 13C NMR (75 MHz, CDCl3) δ/ppm 171.1 (s, 1C), 138.7 (s, 1C), 136.9 (s, 1C), 129.5 (d, 2C), 128.81 (d, 2C), 128.79 (d, 2C), 128.77 (d, 2C), 127.1 (d, 1C), 126.7 (d, 1C), 56.3 (d, 1C), 40.7 (t, 1C), 38.9 (t, 1C), 35.7 (t, 1C), 28.4 (q, 3C); IR (KBr) νmax/cm−1 3343, 3319, 3029, 2986, 2969, 2948, 1685, 1648, 1522, 1372, 1322, 1274, 1232, 1172, 1045, 748, 698, 653. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C22H28N2O3 391.1998; Found 391.1981. Boc-Phe-Phe-(Et-Ph) (47). Prepared from Boc-Phe-Phe-OH (12, 200 mg, 0.49 mmol) and 2-phenylethylamine (67 μL, 0.53 mmol) according to the above general procedure for the peptide coupling via HBTU-HOBT activation. The pure product (207 mg, 83%) was obtained after column chromatography on silica gel by use of HOAc/ EtOAc/CH2Cl2 (1:20:79) as eluent and crystallization from hexane. Colorless solid; mp 78−81 °C; 1H NMR (300 MHz, CD3OD) δ/ppm 7.33−7.10 (m, 15H), 4.52 (t, J = 7.1 Hz, 1H), 4.28−4.16 (m, 1H), 3.44−3.19 (m, 2H), 3.07−3.86 (m, 3H), 2.79−2.61 (m, 3H), 1.35 (s, 9H); 13C NMR (150 MHz, CD3OD) δ/ppm 173.9 (s, 1C), 172.9 (s, 1C), 140.3 (s, 1C), 138.5 (s, 1C), 138.2 (s, 1C), 130.4 (d, 2C), 130.3 (d, 2C), 129.8 (d, 2C), 129.5 (d, 4C), 129.4 (d, 2C), 127.9 (d, 1C), 127.7 (d, 1C), 127.4 (d, 1C), 57.7 (d, 1C), 55.9 (d, 1C), 42.1 (t, 1C), 42.0 (s, 1C), 39.01 (t, 1C), 38.99 (t, 1C), 36.4 (t, 1C), 28.7 (q, 3C), one singlet was not observed; IR (KBr) νmax/cm−1 3298, 3041, 2953, 2930, 1677, 1650, 1220, 1145, 710. HRMS (MALDI-TOF-TOF) m/ z: [M + Na]+ Calcd for C31H37N3O4 538.2682; Found 538.2698. TFA×H-Phe-(Et-Ph) (46). Prepared according to the general procedure for the Boc deprotection from 45 (200 mg, 0.54 mmol) in 83% yield; 172 mg (83%); colorless solid; mp 104−106 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.38−7.14 (m, 10H), 3.96 (dd, J = 7.0, 7.7 Hz, 1H), 3.52−3.45 (m, 1H), 3.36−3.30 (m, 1H), 3.09 (dd, J = 6.2, 5.8 Hz, 1H), 2.98 (dd, J = 6.2, 7.8 Hz, 1H), 2.75−2.66 (m, 2H); 13C NMR (150 MHz, CD3OD) δ/ppm 169.5 (s, 1C), 140.1 (s, 1C), 135.7 (s, 1C), 130.5 (d, 2C), 130.1 (d, 2C), 129.7 (d, 2C), 129.6 (d, 2C), 128.9 (d, 1C), 127.5 (d, 1C), 55.8 (d, 1C), 42.1 (t, 1C), 38.8 (t, 1C), 36.3 (t, 1C); IR (KBr) νmax/cm−1 3402, 3304, 3071, 2928, 1670, 1379, 1204, 1137, 840, 800, 748, 723, 700. HRMS (MALDITOF-TOF) m/z: [M + Na]+ Calcd for C19H19F3N2O2 387.1296; Found 387.1290. TFA×H-Phe-Phe-(Et-Ph) (48). Prepared according to the general procedure for the Boc deprotection from 47 (190 mg, 0.37 mmol) in 63% yield; 127 mg (63%); colorless solid; mp 195−198 °C; 1H NMR (600 MHz, CD3OD) δ/ppm 7.38−7.10 (m, 15H), 4.56 (t, J = 7.6 Hz, 1H), 4.07 (dd, J = 5.6, 8.3 Hz, 1H), 3.43−3.16 (m, 3H), 3.07−2.86 (m, 3H), 2.74−2.60 (m, 2H); 13C NMR (150 MHz, CD3OD) δ/ppm 172.6 (s, 1C), 169.4 (s, 1C), 140.3 (s, 1C), 138.1 (s, 1C), 135.5 (s, 1C), 130.5 (d, 2C), 130.3 (d, 2C), 130.1 (d, 2C), 129.8 (d, 2C), 129.6 (d, 2C), 129.5 (d, 2C), 128.9 (d, 1C), 128.0 (d, 1C), 127.4 (d, 1C), 56.5 (d, 1C), 55.5 (d, 1C), 42.0 (t, 1C), 39.3 (t, 1C), 38.6 (t, 1C), 36.4 (t, 1C); IR (KBr) νmax/cm−1 3285, 3027, 2942, 2926, 1668, 1650, 1561, 1199, 1137, 703. HRMS (MALDI-TOF-TOF) m/z: [M + Na]+ Calcd for C28H28F3N3O3 512.2161; Found 512.2140. PhthAd-Gly-Phe-(Et-Ph) (39). Prepared from 21 (110 mg, 0.29 mmol) and TFA×H-Phe-(Et-Ph) (46, 100 mg, 0.26 mmol) according to the above general procedure for the peptide coupling via HBTUHOBT activation. The pure product (128 mg, 77%) was obtained after column chromatography on silica gel by use of 30% EtOAc/ CH2Cl2 as eluent and increasing polarity to 5% CH3OH/CH2Cl2. Colorless solid; mp 87−91 °C; 1H NMR (600 MHz, CDCl3) δ/ppm 7.76−7.72 (m, 2H), 7.69−7.64 (m, 2H), 7.29−7.14 (m, 8H), 7.07 (d, J = 7.3 Hz, 2H), 6.59 (br. s, 1H), 6.40 (s, 1H), 6.02 (br. s, 1H), 4.57 (dd, J = 7.5, 14.0 Hz, 1H), 3.83 (d, J = 5.2 Hz, 1H), 3.48−3.36 (m, 2H), 3.15 (dd, J = 5.9, 13.8 Hz, 1H), 2.97 (dd, J = 7.4, 13.7 Hz, 1H), 2.75−2.69 (m, 1H), 2.67−2.62 (m, 1H), 2.60−2.50 (m, 4H), 2.49− 2.43 (m, 2H), 2.31−2.27 (m, 2H), 1.88−1.75 (m, 5H), 1.68−1.62 (m, 1H); 13C NMR (150 MHz, CDCl3) δ/ppm 177.6 (s, 1C), 170.4 14919


Article


Irradiation of Peptides in Acetone−D2O in the Presence K2CO3. Peptide 1 (3.23 mg, 4.8 × 10−3 mmol) or 2 (3.16 mg, 3.8 × 10−3 mmol) was dissolved in acetone (2.25 mL) and pored to quartz test tubes. To each test tube a solution of K2CO3 (c = 3.82 × 10−3 M) in D2O or H2O was added (0.63 mL for 1, 0.50 for 2). The samples were purged with N2 (20 min) and irradiated over 6 min in a photoreactor at 300 nm. After the irradiation, H2O was added (5 mL), acetone was removed on a rotary evaporator, and the aqueous mixture was extracted with EtOAc (3 × 5 mL). The solvent from the extracts was removed on a rotary evaporator, and the residue was analized by 1H NMR and HPLC-MS. 1H NMR did not reveal deuterium in the samples, whereas MS indicated 21% D in 33 and 7% D in 34. Irradiation of Peptides in Acetone−D2O in the Presence of Phosphate Buffer. Peptide 1 (6.50 mg, 9.6 × 10−3 mmol) or 2 (6.5 mg, 7.9 × 10−3 mmol) was dissolved in acetone (8 mL) and pored to quartz test tubes. To each test tube a solution of a buffer (2 mL, pH 6.8, K2HPO4−KH2PO4, 50 × 10−3 M) in D2O or H2O was added. The samples were purged with N2 (20 min) and irradiated over 4 min in a photoreactor at 300 nm. After the irradiation, H2O was added (5 mL), acetone was removed on a rotary evaporator, and the aqueous mixture was extracted with EtOAc (3 × 5 mL). The solvent from the extracts was removed on a rotary evaporator, and the residue was analyzed by 1H NMR and HPLC-MS. 1H NMR did not reveal deuterium in the samples, whereas MS indicated 19% D in 33 and 17% D in 34. Investigation of Epimerization in Cyclic Peptides. Two NMR tubes were filled with a solution of 33 (2.23 mg, 3.52 × 10−3 mmol) or 34 (3.69 mg, 4.72 × 10−3 mmol) in d6-acetone (0.46 or 0.62 mL, respectively). To each sample a solution of K2CO3 in D2O was added (c = 3.82 × 10−3 mmol/mL; 0.46 mL for 33 and 0.62 mL for 34). 1H NMR spectra were taken immediately, after 24 h, and after 48 h. In addition, after 48 h, each sample was mixed with H2O (5 mL) and extracted with CH2Cl2 (5 mL) and EtOAc (2 × 5 mL). The solvent from the extracts was removed on a rotary evaporator, and the residue was analyzed by HPLC-MS. Epimerization of samples did not take place, and the samples did not contain any D. Molecular Dynamics (MD) Simulations. We performed 500 ns molecular dynamics (MD) simulations of potassium salts of tetrapeptide 1 and pentapeptide 2 in water. In addition, we performed 500 ns MD simulations of the cyclic tetrapeptide 33 in chloroform and pentapeptide 34 in acetonitrile that are the corresponding solvents in which NOESY experiments were performed. The peptides 1 and 2 were solvated in ca. 1400 TIP3P45 water molecules. The cyclic peptide 33 was solvated in ca. 200 chloroform molecules, whereas the cyclic peptide 34 was solvated in ca. 300 acetonitrile molecules. Unit cell sizes were slightly different in all of the cases, and their size was approximately 35 Å × 35 Å × 35 Å. Peptides and missing residues were described with the GAFF force field.46 For chloroform, we used the all-atom force field,47 and for acetonitrile, we employed the united-atom force field.48 K+ counterion was described with the force field developed by Joung and Cheatham.49 Before production runs, all systems were first minimized and properly equilibrated. MD simulations were performed at a constant temperature of 298 K employing Langevin dynamics with a collision frequency of 2.0 ps−1.50 Pressure was kept at 1 bar using a Berendsen barostat51 and isotropic scaling. 3D periodic boundary conditions were used with long-range electrostatic interactions beyond the nonbonded interaction cutoff of 0.8 nm being accounted for using the particle-mesh Ewald procedure. The SHAKE algorithm52 was employed to constrain all bonds containing hydrogen atoms. Equations of motion were integrated using the leap-from algorithm with a time step of 2 fs. All MD simulations were performed using the program AMBER 16.53

(s, 1C), 169.7 (s, 1C/2C), 168.9 (s, 1C/2C), 138.8 (s, 1C), 136.6 (s, 1C/2C), 134.0 (d, 2C), 132.0 (s, 1C/2C), 129.5 (d, 2C), 128.9 (d, 2C), 128.8 (d, 2C), 128.7 (d, 2C), 127.3 (d, 1C), 126.6 (d, 1C), 122.8 (d, 2C), 60.3 (s, 1C), 54.7 (d, 1C), 43.6 (t, 1C), 42.9 (s, 1C), 41.2 (t, 1C), 40.9 (t, 1C), 39.4 (t, 2C), 38.2 (t, 1C), 38.0 (t, 2C), 35.6 (t, 1C), 35.2 (t, 1C), 29.5 (d, 2C); IR (KBr) νmax/cm−1 3310, 2915, 1857, 1708, 1646, 1524, 1367, 1314, 1076, 717, 699, 642. HRMS (nano-UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C38H40N4O5 633.3077; Found 633.3085. [α]D20 +45.3 (c 0.287, 10% CH3OH/ CH2Cl2). PhthAd-Gly-Phe-Phe-(Et-Ph) (40). Prepared from 21 (200 mg, 0.18 mmol) and TFA×H-Phe-Phe-(Et-Ph) (48, 100 mg, 0.18 mmol) according to the above general procedure for the peptide coupling via HBTU-HOBT activation. The pure product (68 mg, 48%) was obtained after column chromatography on silica gel by use of 0−10% EtOAc/CH2Cl2, followed by preparative TLC using HOAc/CH3OH/ CH2Cl2 (1:10:89) as eluent. Colorless solid; mp 84−86 °C; 1H NMR (300 MHz, CDCl3) δ/ppm 7.70−7.55 (m, 5H), 7.37 (d, J = 8.6 Hz, 1H), 7.20−6.96 (m, 16H), 6.66 (br. s, 1H), 4.78 (dd, J = 7.4, 15.4 Hz, 1H), 4.68 (dd, J = 6.6, 13.5 Hz, 1H), 3.96−3.73 (m, 2H), 3.43−3.19 (m, 2H), 2.95−2.77 (m, 4H), 2.61 (t, J = 7.4 Hz, 2H), 2.54−2.35 (m, 6H), 2.23 (br. s, 2H), 1.84−1.52 (m, 6H); 13C NMR (75 MHz, CDCl3) δ/ppm 177.1 (s, 1C), 171.1 (s, 1C), 170.5 (s, 1C), 169.6 (s, 2C), 168.7 (s, 1C), 139.1 (s, 1C), 136.7 (s, 1C/2C), 136.3 (s, 1C/ 2C), 133.9 (d, 2C),131.9 (s, 1C), 129.5 (d, 2C), 129.4 (d, 2C), 128.8 (d, 2C), 128.6 (d, 2C), 128.54 (d, 2C), 128.47 (d, 2C), 127.0 (d, 1C), 126.8 (d, 1C), 126.4 (d, 1C), 122.8 (d, 2C), 60.3 (s, 1C), 54.61 (d, 1C), 54.56 (d, 1C), 43.4 (s, 1C), 42.8 (t, 1C), 41.3 (t, 1C), 41.1 (t, 1C), 39.4 (t, 1C), 39.3 (t, 1C), 39.2 (t, 1C), 38.1 (t, 1C), 38.0 (t, 1C), 35.6 (t, 1C), 35.3 (t, 1C), 33.9 (t, 1C), 29.6 (d, 2C); IR (KBr) νmax/cm−1 3300, 3060, 3028, 2918, 1856, 1710, 1638, 1527, 1368, 1314, 1079, 718, 698. HRMS (nano-UPLC-ESI-qTOF) m/z: [M + H]+ Calcd for C47H49N5O6 780.3761; Found 780.3773. [α]D20 +67.2 (c 0.253, 10% CH3OH/CH2Cl2). Photochemical Reaction Quantum Yield Determination. The quantum yield of photocyclization of peptides 1−6 in CH3CN was determined by use of a KI/KIO3 actinometer. For the actinometer, a 0.1 M solution of KIO3 in borate buffer (0.01 M, pH 9.25) was prepared, which was used to prepare a fresh 0.6 M KI solution in borate buffer of KIO3. In quartz cuvettes, fresh solutions of 1−6 (c ≈ 2 × 10−4 M, 2.5 mL) in CH3CN−H2O (1:3) in the presence of potassium phosphate buffer (c = 50 mM, pH = 6.0) were prepared. Prior to the irradiations, UV−vis spectra were recorded and the solutions were purged with Ar for 30 min. The solutions were irradiated in a Luzchem reactor with one lamp at 254 nm. UV−vis spectra were recorded after 0, 0.5, 1, and 1.5 min irradiation for the actinometer, and HPLC analyses were performed after 30, 60, and 90 min irradiation for compounds 1−6. The quantum yield of photocyclization for peptides 1−6 in acetone was determined by use of a secondary actinometer, 4-[(Nphthalimido)methyl]cyclohexane carboxylic acid (Φ = 0.30).33 In quartz test tubes, fresh solutions of 1−6 (c = 2 × 10−4 M, 10 mL) in acetone−H2O (1:1) in the presence of potassium phosphate buffer (c = 50 mM, pH = 6.0) were prepared. The solutions were purged with Ar for 30 min and then irradiated in a Luzchem reactor with 4 lamps at 300 nm for 3 min. HPLC analyses of the irradiated solutions were performed after 0, 1, and 3 min irradiation. All measurements were done in triplicate, and the average value was reported. Equations to calculate Φ can be found in the Supporting Information. Laser Flash Photolysis (LFP). All LFP studies were performed on a system previously described43 using as an excitation source a pulsed Nd:YAG laser at 266 nm (

Photocyclization of Tetra- and Pentapeptides Containing

Recommend Documents