Interplay Of Stereochemistry, Conformational Rigidity, And Ease Of

Research Article pubs.acs.org/acscombsci

Interplay Of Stereochemistry, Conformational Rigidity, And Ease Of Synthesis For 13-Membered Cyclic Peptidomimetics Containing APC Residues Dongyue Xin, Andrew Jeffries, and Kevin Burgess* Department of Chemistry, Texas A & M University, Box 30012, College Station, Texas 77842, United States S Supporting Information *

ABSTRACT: As part of a program to design small molecules that bind proteins, we require cyclic peptides (or peptidomimetics) that are severely constrained such that they adopt one predominant conformation in solution. This paper describes syntheses of the 13-membered cyclic tetrapeptides 1 containing aminopyrrolidine carboxyl (APC) residues. A linear precursor was prepared and used to determine optimal conditions for cyclization of that substrate. A special linker was prepared to enable cyclization of similar linear peptidomimetics on a solid phase, and the solution-phase cyclization conditions were shown to be appropriate for this too. Stereochemical variations were then used to determine the ideal APC configuration for cyclization of the linear precursors (on a solid phase, using the conditions identified previously). Consequently, a series of compounds were prepared that are representative of compounds 1. Conformational studies of representative compounds in DMSO solution were performed primarily using (i) NOE studies, (ii) quenched molecular dynamics simulations using no constraints from experiment, and (iii) MacroModel calculations with NMR constraints. All three strategies converged to the same conclusion: the backbone of molecules based on 1 tends to adopt one preferential conformation in solution and that conformation can be predicted from the stereochemistries of the αamino acids involved. KEYWORDS: peptidomimetics, cyclic peptide, conformational studies, quenched molecular dynamics, macromdel calculations

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α- with a cyclic β-amino acid in a linear peptide mimic extends the main chain by one atom but does not seriously compromise its rigidity because the original residue and its replacement both have two significant degrees of freedom.

INTRODUCTION Amide bonds in peptide conformations are presumed to be planar and s-trans, so a Gly residue may be said to have only two significant degrees of freedom, corresponding to its ϕ and ψ dihedral angles. Rotational impediments on ϕ and ψ dihedral angles imposed by side-chains mean that all the other genetically encoded residues are more constrained than Gly. Nevertheless, there is significant flexibility about ϕ and ψ dihedral angles for all the genetically encoded amino acids except proline (that is more constrained); hence, they may be regarded as having two significant degrees of freedom.

Peptidomimetics from acyclic β-amino acids will almost invariably be more flexible than the corresponding ones in genetically encoded peptides because they have three significant degrees of freedom. However, like proline, cyclic β-amino acids are less flexible than their acyclic analogs: they have only two significant degrees of freedom. Consequently, substitution of an © XXXX American Chemical Society

Received: March 1, 2017 Revised: April 10, 2017

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DOI: 10.1021/acscombsci.7b00041 ACS Comb. Sci. XXXX, XXX, XXX−XXX

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ACS Combinatorial Science

favoring closure to 13-membered systems. Consequently, the research described here features the first reported syntheses of cyclic peptidomimetics containing APC residues, specifically the 13-membered ring derivatives 1.

On the basis of the generalities outlined above, it is to be expected that substitution of α-amino acids in peptides with cyclic β-amino acids tends to extend them without significantly increasing their flexibility. However, syntheses of substituted carbocyclic β-amino acids are nontrivial so it is difficult to preserve side-chain functionalities when α-amino acids are substituted with cyclic β-amino acids. Aminopyrrolidine carboxyl (APC) residues like A and B are special because they are relatively rigid and have an endocyclic N-atom that could be alkylated with groups to mimic sidechains or serve other purposes. Gellman’s group was first to use APC residues A and ent-A in linear peptidomimetics.1,2 To facilitate this, they devised a synthesis of A and ent-A that involved an inexpensive chiral auxiliary (1-phenylethylamine, available as both enantiomers) and purification of diastereomers via recrystallization.3,4 Consequently, there are at least 16 papers in the literature that report applications of A and entA in foldamers.5−7 For instance, these residues have been incorporated into peptidomimetics intended to disrupt protein−protein interactions (PPIs, e.g., in BH3 domains,8−10 glucagon-like peptide 1,11 vascular endothelial growth factor12), as scaffolds for presenting carbohydrates13 or peptide nucleic acid residues,14 and as mimics of nonhelical secondary structures.15

Targets 1 were selected because they have several attractive features. We hypothesized macrocycles 1 may adopt single conformational states in solution; that rigidity is useful when attempting to understand how preferred solution state conformations relate to binding. Unpublished molecular dynamics calculations from our group that suggest 12membered ring cyclic tetrapeptides also should be conformationally rigid provided they do not contain Gly and Pro residues, but these are hard to prepare. Conversely, we suspected compounds 1 might be far more accessible via solid phase syntheses than cyclic tetrapeptides composed of only α-amino acids. These putative attributes, and the fact that the pyrrolidine NH could be functionalized, are valuable to our broader research program that features conformationally rigid compounds that can present three amino acid side-chains in disruption of protein−protein interactions.34,35 Consequently, we decided to study synthetic accessibilities and the conformational biases of the APC-containing, cyclic tetrapeptides 1. It emerges here that the featured compounds 1 are (i) accessible via solid phase syntheses, (ii) constrained to single conformers in DMSO solution, and unexpectedly, (iii) are inclined to adopt conformations that correlate with the stereochemistries of their constituent amino acids in a predictable way.

The cis-APC stereomers B are more time-consuming to prepare in enantiomerically pure form than their trans-isomers A,16−18 hence it is unsurprising that they have been used less frequently. Most of the literature on cis-APCs B and ent-B concerns specific applications in medicinal chemistry,19−24 but there are few in peptidomimetics. A long-term project in our laboratories requires conformationally rigid cyclic peptidomimetics that can be prepared expeditiously via solid phase methods. To our surprise, we were unable to find examples of cyclic peptidomimetics containing either trans- or cis-APC residues. Broadening the search to include peptidomimetics containing carbocyclic analogs of APCs like C and D also revealed little information. There is one report of a cyclic peptidomimetic based on C25 and one on D;26 in each case these are larger, and presumably more conformationally flexible, macrocycles than the ones in this manuscript (see below).

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RESULTS AND DISCUSSION Syntheses. Samples of the trans-APC compounds required in this study were prepared via Gellman’s method.3 Syntheses of their cis-isomers were achieved via a small modification of a literature procedure by Confalone et al.;16 this change enabled access to the hitherto unknown FMOC-protected compounds 3 and 4 (Scheme 1). Microwave (mw) assisted solid phase syntheses of the linear APC-containing peptidomimetics 5 were performed as outlined in Scheme 2. Use of chlorotrityl resin (Chem-Impex) gave relatively hindered esters that we hypothesized would protect the APC residues from epimerization.36 In the event, the overall procedure worked well to give the desired linear products in >95% crude purity by HPLC (UV detection at 254 nm), and no epimerization was detected. The sequence LLL-5aay′ was used for optimization studies (throughout this paper lower case one-letter codes relate the side-chains R1−R3 to the closest amino acid, and primed letters denote protection, e.g., y′ for −CH2C6H4−4-OtBu and a for −CH3 of Ala). In fact, the calculations described below were performed for the sequence LLL-5aaa but it was impractical to focus on that motif for experimental studies because Ala-AlaAla has no conspicuous chromophore; consequently, we settled on LLL-5aay′ for optimization. Approximately 20 sets of conditions were tested for cyclization of (R,S)-APC-LLL-5aay′

12-Membered cyclic tetrapeptides are notoriously difficult to prepare,27−33 but we suspected replacement of a genetically encoded amino acid with an APC residue should enhance the conformational rigidity (amino acid with 2 significant degrees of freedom replaced by another having the same, see above) while the extra atom could reduce ring strain effects thus B


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ACS Combinatorial Science Scheme 1. Synthesis of New, FMOC-Protected, cis-APC Derivatives

(Figure 1) and the conditions shown in Figure 1a were the best identified for this cyclization step.

Figure 1. Cyclizations of LLL- and DDD-stereoisomers of the linear peptidomimetics to give protected forms of the featured product 6.

Scheme 2. Solid-Phase Syntheses of Linear Peptidomimetics 5

Having found suitable conditions for cyclization of (R,S)APC-LLL-5aay′, we set out to determine the APC stereoisomer that would best facilitate cyclization of LLL-5aay′. Compound availability issues made it more convenient to test (R,R)-APCDDD-5aay′ than (S,S)-APC-LLL-5aay′ and (R,S)-APC-DDD5aay′ than (S,R)-APC-LLL-5aay′, but in each case the result would be the same since these are pairs of enantiomers. Thus, the compounds in Figure 1b represent one enantiomeric form of every energetically distinct cyclization via the disconnection implied in Figure 1. In the event, the cis-APC, (R,R)-APCDDD-6aay′ gave a higher yield of its corresponding cyclization product than any of the other reactions. Undesired cyclic dimer formed in all four reactions (LC-MS), but least for the cisisomer (R,R)-APC-DDD-6aay′. These observations indicate cyclization to the enantiomer of (R,R)-APC-DDD-6aay′, and (S,S)-APC-LLL-6aay′, would be similarly favored. Thus, cis(S,S)-APC was more conducive to efficient cyclization than either of the trans-isomers when the α-amino acids were in the L-configuration. It is for that reason that only cis-(S,S)-APC was used in subsequent studies. Cyclization of all isomers of (S,S)-APC-6aay′ were studied to explore if these reactions can be as efficient as those described above but when other α-amino acid stereomers are used. Figure 2b indicates that the least efficient cyclization to the desired monomeric cyclic peptidomimetic was for the (S,S)-APC combination with DDD-6aay′. Close examination of the data in Figure 2b reveals that the cyclization reactions are more efficient when the N-terminal residue AA1 has the Lconfiguration than in comparable reactions in which it has Dstereochemistry. This assertion is true for all the examples shown in Figure 2b (consider the pairs connected by a bracket on the right side of that graphic). Figure 2c summarizes isolated yields (based on resin loading) for formation of products 1 after solid-phase syntheses of the C


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Scheme 3. Synthesis of a cis-APC-Loaded Linker for OnBead Cyclization

conditions (Scheme 4). A problem was encountered for the FMOC-deprotection step; specifically, 2% DBU in dioxane (conditions we had used previously to remove FMOC without cleavage of the nosyl-like linker)37,38 caused epimerization adjacent the APC-allyl ester. In response, 20% piperidine in DMF at room temperature for 3 min was used for the FMOC deprotection. When this reaction was performed at elevated temperatures or for longer times then partial cleavage of the nosyl-like linker was observed. Scheme 4 showed Pd-mediated cleavage of the allyl ester then the final FMOC deprotection before the critical on-bead cyclization. If the order of these steps was reversed then impurities formed, probably because of allylation of the Nterminal amine. The cyclization step was performed using similar conditions to those that had been developed for solution (Figures 1 and 2); the only changes that were made were to use a less expensive carbodiimide, and CH2Cl2/DMF because the reaction on TentaGel was faster and gave higher purities in that medium. Conformational Analyses. The following approach was used to investigate conformations of the target cyclic peptidomimetics 1. First a molecular dynamics technique (quenched molecular dynamics, QMD39,40) was used to ascertain (i) the most favorable conformation that could be found and (ii) the diversity of simulated conformations in a continuous medium of dielectric 46.7 (corresponding to DMSO). As mentioned above, these calculations were performed on (S,S)-APC-LLL-1aaa; this gives the calculated, preferred conformations of the backbone with only the simplest possible side-chains.

Figure 2. Cyclization a complete set of stereoisomers to afford the protected macrocycles 6. (a) Reaction conditions, (b) HPLC yield vs time plots, and (c) summary of isolated yields for cyclic products 1 based on loading of the resin and after rpHPLC purification.

linear precursors, cleavage from the resin, cyclization in solution under dilute conditions, then deprotection of tBu/BOC sidechain and the pyrrolidine-N. The sequence involves many steps (four coupling/deprotection reactions, cleavage, cyclization, and deprotection) so these yields indicate the process is highly efficient overall. In the experiments described above, it was advantageous to break the synthesis down into on-resin preparation of linear peptides then cyclization. However, once the process had been optimized, an approach using on-bead cyclization was desired for accelerated production of the peptidomimetics 1, hence a linker that was stable to acid and base was necessary to realize that approach. Scheme 3 shows how a nosyl-based system developed in our laboratories37,38 was modified to fulfill these needs. The known compound E was hydrolyzed to 7 then coupled with Gly. Oxidation of sulfide 8 to the sulfonyl chloride 9 was achieved using a chlorination reagent (1,3-dichloro-5,5dimethylhydantoin) then the featured FMOC-protected cis(S,S)-APC 4 was added and the tBu group was removed to give the completed “handle” 10. Solid-phase syntheses of the cyclic products 1 began with coupling handle 10 to TentaGel-NH2 resin under microwave D


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using Macromodel41 with NMR constraints (distance limitations from NOESY). Using two simulation approaches in this way is advantageous because they are complementary: confidence in the calculations is increased if the conformations deduced without using experimental data (QMD) compare well with those that employ NMR constraints (Macromodel). Moreover, strategies using NMR constraints are not necessarily more realistic than unconstrained calculations because (i) NOE/ROE effects depends on 1/r6 distance relationships,42 hence close contacts are accentuated and distal ones are less conspicuous and (ii) conformations deduced from molecular dynamics calculations constrained in accord with NMR data are less reliable for small molecules (compared with proteins for example) because relatively few data points are involved. Figure 3 shows conformations for one enantiomeric series of compounds (S,S)-APC-LLL-1aaa in black (from QMD) overlaid with the corresponding conformations of (S,S)-APCLLL-1aay in silver (from Macromodel with NOE constraints). There is a maximum RMSD of 0.56 Å (based on Cα−Cβ) indicating a good correspondence between both strategies. This observation indicates a high level of confidence overall in the deduced conformations. One of the strengths of the QMD approach is that it indicates the diversity of conformers that are energetically accessible. Flexible molecules will afford multiple families, each consisting of a cluster of conformations. However, QMD analyses of all the stereoisomers shown in Figure 3 each gave one predominant family. Only some of the stereomers gave two families, and in the cases where there were two, the second family was formed from far fewer conformations, and the lowest energy conformer in that group was higher than the corresponding one in the primary family. These observations indicate the APC-containing molecules 1 are almost conformationally homogeneous in solution. This assertion is supported by the proton NMR data that shows single sets of resonances for each stereoisomer with sharp, resolved peaks for the sidechains and for the amide protons. Review of the structures in Figure 3 shows there are no Hbonded NH atoms in the preferred conformers, and the degree of solvent shielding by the ring is difficult to access. Consistent with this temperature coefficient data43,44 (Table 1) gave no discernible trend. The series of structures featured in Table 1 and Figure 3 have a fixed configuration at the (S,S)-APC residue hence an enantiomer exists for each of the compounds illustrated. Consistent with this, the CD spectra recorded for the set of isomers shown in Figure 4 does not vary in a regular stepwise pattern between the molar elipticity extremes. However, one crude trend is evident: the isomers predominantly consisting of all L-α-amino acids have negative molar elipticity maxima, while those from D-α-amino acids tend to have positive molar ellipticities.

Scheme 4. Solid-Phase Syntheses of the Featured Compounds 1 via On-Bead Cyclization

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CONCLUSION Unlike cyclic tetrapeptides composed of α-amino acids, the 13membered ring systems featured in this work can be prepared efficiently via solution or solid phase methods. Conformational analyses by molecular dynamics showed that the stereochemistry of the APC residues do not have a profound impact on the conformations of the cyclic systems 1. Our experimental data shows that the cyclization efficiencies were significantly influenced by the APC stereochemistry. It is a little unfortunate that the cis-APC isomers are favored because these are more

Data from the QMD experiments was then compared with calculations and experimental NMR data obtained for (S,S)APC-LLL-1aay. This comparison is informative because QMD does not use any constraints f rom experimental data. In parallel, conformations of the cyclic compounds 1 were also determined E


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Table 1. Temperature Coefficient Data for Isomers of (S,S)APC-1aay in DMSO

−Δδ/ΔT (PPB/K in DMSO-d6) diasteromer (S,S)-APC-???-1aay

APC-NH

Ala1-NH

Ala2-NH

Tyr-NH

LLL DLL LDL LLD DDL DLD LDD DDD

1.5 2.3 5.8 3.4 4.2 2.0 3.6 3.6

1.4 0.8 3.6 2.3 3.1 3.3 1.4 2.7

1.7 3.9 5.0 6.6 4.6 4.6 2.0 4.0

2.0 4.0 3.1 0.0 2.7 5.9 1.4 2.1

Figure 4. CD spectra of (S,S)-APC-LLL-1aay in acetonitrile.

time-consuming than the trans-ones to prepare, but overall, this is not a major factor. The CD spectra shown in Figure 4 are informative insofar as they show a steady gradation between extremes (even though the stereochemistry of the APC residue is constant throughout). This implies that the conformations of these molecules vary in a stepwise fashion as the stereochemistry of the α-amino acids is changed. We have observed exactly the same phenomena in a study of 13-membered ring compounds containing anthranilic acid,45 and explain this in a similar way. Calculated structures in Figure 5 indicate the carbonyl groups point downward around the equator of the LLL-isomer, and all upward in the DDD-structure. Isomers representing transitions between these extremes have carbonyl groups that follow this trend, that is, ones that are part of L-amino acids point down, while those on D-amino acids point up. Overall, this paper indicates that it is possible to prepare the targeted 13-membered ring compounds via an efficient ring closure step, yet the products retain a high degree of conformational rigidity. It is an advantage that both solution and solid phase methods have been developed because the former allows for scalable preparation of select compounds while the latter facilitates parallel syntheses of libraries.

Figure 3. Conformations of (S,S)-APC-LLL-1aaa deduced without NMR constraints overlaid with conformations of (S,S)-APC-LLL-1aay from the strategy described with NMR constraints. F



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Moreover, the existence of an easily understood correlation between conformations of the products and the stereochemistries of the α-amino acid building blocks is an attractive feature of this work.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscombsci.7b00041. Experimental procedures and spectroscopic data for all compounds (PDF)

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REFERENCES

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Figure 5. Systematic variation of preferred conformations with αamino acid stereochemistry for (S,S)-APC-1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kevin Burgess: 0000-0001-6597-1842 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this project was provided by the National Science Foundation (CHE-1608009), National Institutes of Health (HL126346), The Robert A. Welch Foundation (A1121), DoD BCRP Breakthrough Award (BC141561), and CPRIT (RP150559 and RP170144). We thank Dr. Lisa M. Perez for useful discussions. NMR instrumentation at Texas A&M University was supported by a grant from the National Science Foundation (DBI-9970232) and the Texas A&M University System. G


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