Felix Freire - Academia.edu (original) (raw)

Papers by Felix Freire

Journal of The American Chemical Society, 2009

Cyclization is a powerful strategy for inducing antiparallel -sheet secondary structure in relati... more Cyclization is a powerful strategy for inducing antiparallel -sheet secondary structure in relatively short peptide segments. Biological examples feature cyclization via the backbone, 1 as seen in gramicidin S and θ-defensin, and cyclization via side chains (disulfide formation), 2 as seen in tachyplesins and protegrins. These natural prototypes have inspired the use of cyclization in -sheet design efforts aimed at both structural and functional goals. For example, both backbone and side chain cyclization have been used to generate peptides that serve as spectroscopic references for the -sheet conformations adopted by flexible, linear peptides. 3 Cyclic -sheet scaffolds have provided a fruitful basis for development of peptides with a variety of biological activities, including antibiotics, vaccine epitopes, RNA ligands, and, perhaps most intriguingly, helixmimetic inhibitors of protein-protein recognition. 4 The demonstrated utility of cyclically enforced antiparallel -sheet scaffolds raises the prospect that analogous parallel -sheet scaffolds would be comparably useful. Biology does not offer a clear basis for achieving this structural goal in relatively small molecules. Interstrand disulfides seem to be incompatible with a parallel -sheet secondary structure, given the rarity of such crosslinks in proteins. 5 -Strand-forming segments must be linked N-terminus-to-C-terminus within peptides and proteins; therefore, covalent connection of -strands in parallel orientation requires a peptidic linker that is at least as long as the -strands themselves. This topological limitation has inspired many efforts to devise small nonpeptide units that can be used to connect peptide segments in a C-to-C or N-to-N fashion. 6-8 Such units should ideally have a strong turn-forming propensity that can encourage -sheet interactions between attached peptide segments. We have developed both preorganized C-to-C linkers and preorganized N-to-N linkers that promote (but do not enforce) parallel -sheet formation between peptide segments in aqueous solution ). 9 Here we evaluate these linkers in the context of backbone cyclization. Unexpectedly, we find that only one of the two turn units needs to be preorganized to enforce a high level of parallel -sheet folding.

Journal of The American Chemical Society, 2008

Short peptides that adopt specific secondary structures in aqueous solution have proven to be inv... more Short peptides that adopt specific secondary structures in aqueous solution have proven to be invaluable tools for elucidating the balance of forces that controls the stability of these common protein substructures. Rules for the design of sequences that fold autonomously into the R-helical conformation have been available for nearly two decades, 1 and the analysis of R-helical folding preferences is now a mature field. Guidelines for the design of antiparallel -sheet-forming sequences have emerged more recently, enabling fundamental studies of the origins of antiparallel -sheet stability. 2 Parallel -sheet is common in proteins, but this structural motif has received little attention to date because the rules necessary to design molecules that will adopt this secondary structure in aqueous solution are not yet fully developed.

Angewandte Chemie-international Edition, 2007

Understanding the factors responsible for the stability of common protein secondary structures ha... more Understanding the factors responsible for the stability of common protein secondary structures has been a longstanding goal. Analysis of b sheets has lagged behind the study of a helices because the development of suitable model systems has been more challenging for the former than for the latter. [1] Over the past decade, however, rules for the design of b sheets that fold autonomously in water have been elucidated, with a particular emphasis on two-stranded antiparallel b sheets ("b hairpins"), which represent a minimum increment of this secondary structure. These model systems have proved useful for evaluating the contributions of factors such as side-chain-side-chain interactions, [4] interstrand linker composition, strand length, and strand number to the stability of b sheets. [7] As an outgrowth of this work, b hairpins have emerged as excellent platforms for the evaluation of noncovalent interactions that do not necessarily occur naturally in b sheets. [8] b Hairpins and hairpin-like molecules stabilized by cyclization are also attractive systems for biomedical applications and fundamental studies. [9] The use of designed peptides to probe the origins of bsheet stability, or to study noncovalent attractions between moieties that become spatially juxtaposed upon folding, requires the ability to determine the extent of b-sheet folding in solution. If only two conformational states are populated, unfolded and b sheet, then determining the population of these two states gives the folding equilibrium constant (K fold ), which provides insight into the stability of the folded state (DG fold = ÀR T lnK fold ). For proteins that adopt defined tertiary structures, conformational stability is often assessed by using heat or a chemical denaturant to disrupt the folded state while monitoring the extent of folding by a conformationally sensitive spectroscopic probe (for example, circular dichroism). This approach is convenient because globular proteins are typically completely folded near room temperature and in the absence of denaturant ("native conditions"), which establishes the spectroscopic signature of the folded state, and because it is often straightforward to identify the spectroscopic signature of a fully unfolded state generated at high temperature or high denaturant concentration. In contrast, most of the autonomously folding b-sheet model systems described to date cannot be driven by changing the conditions to the limiting states. Therefore, identifying the spectroscopic signatures for the fully unfolded and fully folded states of b-sheet model systems has frequently required the preparation and characterization of distinct reference peptides or the implementation of elaborate data analysis techniques. [4][7][8] We have recently developed a new approach for studying the conformational stability of small proteins, [11] and here we describe the extension of this approach to b hairpins. This method involves polypeptide analogues in which one backbone amide group has been replaced by a thioester (namely, thiodepsipeptides). The conformational stability is assessed by monitoring the equilibrium constant for a thiol-thioester exchange reaction that causes the full-length thiodepsipeptide to be reversibly cleaved, which precludes adoption of a native-like fold. The "backbone thioester exchange" (BTE) measurements can be conducted under native conditions, and it is not necessary for the full-length molecule to be completely folded under these conditions. Our previous BTE studies have focused on polypeptides that adopt a discrete tertiary structure. [11] Here we show that the BTE method can be extended to a secondary structure model system ).

Angewandte Chemie, 2007

Understanding the factors responsible for the stability of common protein secondary structures ha... more Understanding the factors responsible for the stability of common protein secondary structures has been a longstanding goal. Analysis of b sheets has lagged behind the study of a helices because the development of suitable model systems has been more challenging for the former than for the latter. [1] Over the past decade, however, rules for the design of b sheets that fold autonomously in water have been elucidated, with a particular emphasis on two-stranded antiparallel b sheets ("b hairpins"), which represent a minimum increment of this secondary structure. These model systems have proved useful for evaluating the contributions of factors such as side-chain-side-chain interactions, [4] interstrand linker composition, strand length, and strand number to the stability of b sheets. [7] As an outgrowth of this work, b hairpins have emerged as excellent platforms for the evaluation of noncovalent interactions that do not necessarily occur naturally in b sheets. [8] b Hairpins and hairpin-like molecules stabilized by cyclization are also attractive systems for biomedical applications and fundamental studies. [9] The use of designed peptides to probe the origins of bsheet stability, or to study noncovalent attractions between moieties that become spatially juxtaposed upon folding, requires the ability to determine the extent of b-sheet folding in solution. If only two conformational states are populated, unfolded and b sheet, then determining the population of these two states gives the folding equilibrium constant (K fold ), which provides insight into the stability of the folded state (DG fold = ÀR T lnK fold ). For proteins that adopt defined tertiary structures, conformational stability is often assessed by using heat or a chemical denaturant to disrupt the folded state while monitoring the extent of folding by a conformationally sensitive spectroscopic probe (for example, circular dichroism). This approach is convenient because globular proteins are typically completely folded near room temperature and in the absence of denaturant ("native conditions"), which establishes the spectroscopic signature of the folded state, and because it is often straightforward to identify the spectroscopic signature of a fully unfolded state generated at high temperature or high denaturant concentration. In contrast, most of the autonomously folding b-sheet model systems described to date cannot be driven by changing the conditions to the limiting states. Therefore, identifying the spectroscopic signatures for the fully unfolded and fully folded states of b-sheet model systems has frequently required the preparation and characterization of distinct reference peptides or the implementation of elaborate data analysis techniques. [4][7][8] We have recently developed a new approach for studying the conformational stability of small proteins, [11] and here we describe the extension of this approach to b hairpins. This method involves polypeptide analogues in which one backbone amide group has been replaced by a thioester (namely, thiodepsipeptides). The conformational stability is assessed by monitoring the equilibrium constant for a thiol-thioester exchange reaction that causes the full-length thiodepsipeptide to be reversibly cleaved, which precludes adoption of a native-like fold. The "backbone thioester exchange" (BTE) measurements can be conducted under native conditions, and it is not necessary for the full-length molecule to be completely folded under these conditions. Our previous BTE studies have focused on polypeptides that adopt a discrete tertiary structure. [11] Here we show that the BTE method can be extended to a secondary structure model system ).

Journal of The American Chemical Society, 2007

Aminoglycosides are clinically relevant antibiotics that participate in a large variety of molecu... more Aminoglycosides are clinically relevant antibiotics that participate in a large variety of molecular recognition processes involving different RNA and protein receptors. The 3-D structures of these policationic oligosaccharides play a key role in RNA binding and therefore determine their biological activity. Herein, we show that the particular NH2/NH3 + /OH distribution within the antibiotic scaffold modulates the oligosaccharide conformation and flexibility. In particular, those polar groups flanking the glycosidic linkages have a significant influence on the antibiotic structure. A careful NMR/theoretical analysis of different natural aminoglycosides, their fragments, and synthetic derivatives proves that both hydrogen bonding and chargecharge repulsive interactions are at the origin of this effect. Current strategies to obtain new aminoglycoside derivatives are mainly focused on the optimization of the direct ligand/receptor contacts. Our results strongly suggest that the particular location of the NH 2/NH3 + /OH groups within the antibiotics can also modulate their RNA binding properties by affecting the conformational preferences and inherent flexibility of these drugs. This fact should also be carefully considered in the design of new antibiotics with improved activity.

Chemical Communications, 2007

A complete characterisation of the protonation equilibrium that accompanies the molecular recogni... more A complete characterisation of the protonation equilibrium that accompanies the molecular recognition of neomycin-B by a specific RNA receptor has been achieved by employing simple NMR measurements.

Chemistry-a European Journal, 2005

The specific interaction of a variety of modified hevein domains to chitooligosaccharides has bee... more The specific interaction of a variety of modified hevein domains to chitooligosaccharides has been studied by NMR spectroscopy in order to assess the importance of aromatic–carbohydrate interactions for the molecular recognition of neutral sugars. These mutant AcAMP2-like peptides, which have 4-fluoro-phenylalanine, tryptophan, or 2-naphthylalanine at the key interacting positions, have been prepared by solid-phase synthesis. Their three-dimensional structures, when bound to the chitin-derived trisaccharide, have been deduced by NMR spectroscopy. By using DYANA and restrained molecular dynamics simulations with the AMBER 5.0 force field, the three-dimensional structures of the protein–sugar complexes have been obtained. The thermodynamic analysis of the interactions that occur upon complex formation have also been carried out. Regarding binding affinity, the obtained data have permitted the deduction that the larger the aromatic group, the higher the association constant and the binding enthalpy. In all cases, entropy opposes binding. In contrast, deactivation of the aromatic rings by attaching fluorine atoms decreases the binding affinity, with a concomitant decrease in enthalpy. The role of the chemical nature of the aromatic ring for establishing sugar contacts has been thus evaluated.

Chemical Communications, 2008

A simple NMR methodology, through the formation of chiral BINOL borates in the NMR tube, and that... more A simple NMR methodology, through the formation of chiral BINOL borates in the NMR tube, and that reunites the advantages of chiral derivatizing (CDAs) and chiral solvating agents (CSAs), is presented for the assignment of the absolute configuration of aand b-hydroxy acids.

Organic Letters, 2006

The absolute configuration of 1,2,3-prim,sec,sec-triols can be assigned by comparison of the 1 H ... more The absolute configuration of 1,2,3-prim,sec,sec-triols can be assigned by comparison of the 1 H NMR spectra of the tris-(R)-and the tris-(S)-MPA ester derivatives. An experimental demonstration of this correlation with 24 triols of known absolute configuration and a protocol using two parameterss∆δ RS (H3) and the difference between ∆δ RS (H2) and ∆δ RS (H3) ) |∆(∆δ RS )|sfor its application to the determination of the absolute configuration of other triols are presented.

Cheminform, 2006

The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperatu... more The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperature NMR of a bis-MPA ester derivative. The assignment requires the analysis of just the methylene protons, is not limited by the absence of signals from the R group of the diol, and requires a very small and recoverable sample.

Chemical Communications, 2005

Chemistry-a European Journal, 2009

The conformational compositions of the tris(α-methoxy-α-phenylacetic acid) ester derivatives of 1... more The conformational compositions of the tris(α-methoxy-α-phenylacetic acid) ester derivatives of 1,2,3-prim,sec,sec-triols are presented. These conformations have been determined by theoretical and experimental data (i.e., energy- and chemical-shift calculations, circular dichroism (CD) experiments, coupling-constant analysis, enantioselective deuteration experiments, and low-temperature NMR spectroscopic studies). A detailed analysis of the anisotropic effects due to the most significant conformers in the 1H NMR spectra supported the correlation between the 1H NMR spectra (ΔδRS value of H(3′) and |Δ(ΔδRS)| parameters) and the absolute configuration of the substrate. The study also allows the identification of the pro-R and pro-S methylene protons from their vicinal coupling constants and relative chemical shifts.

Chemistry-a European Journal, 2005

The absolute configuration of 1,2-diols formed by a primary and a secondary (chiral) hydroxyl gro... more The absolute configuration of 1,2-diols formed by a primary and a secondary (chiral) hydroxyl group can be deduced by comparison of the 1H NMR spectra of the corresponding (R)- and bis-(S)-MPA esters (MPA=methoxyphenylacetic acid). This method involves the use of the chemical shifts of substituents L1/L2 attached to the secondary (chiral) carbon, and of the hydrogen atom linked to the chiral center (CαH) as diagnostic signals. Theoretical (AM1, HF, and B3 LYP calculations) and experimental data (dynamic and low-temperature NMR spectroscopy, studies on deuterated derivatives, constant coupling analysis, circular dichroism (CD) spectra, and NMR studies with a number of diols of known absolute configuration) prove that the signs of the ΔδRS obtained for those signals correlate with the absolute configuration of the diol. A graphical model for the reliable assignment of the absolute configuration of a 1,2-diol by comparison of the NMR spectra of its bis-(R)- and bis-(S)-MPA esters is presented.

Organic Letters, 2010

The absolute configuration of a 1,2-primary/secondary diol can be easily determined by preparatio... more The absolute configuration of a 1,2-primary/secondary diol can be easily determined by preparation of its bis-(R)-and bis-(S)-9-AMA ester derivatives, followed by comparison of the NMR chemical shifts of the diastereotopic methylene protons in the two derivatives. Alternatively, the assignment can be carried out using only one derivative if the evolution with temperature of the signals corresponding to the CrH protons is analyzed.

Journal of Organic Chemistry, 2007

Comparison of the room-and low-temperature 1 H NMR spectra of the bis-(R)-or bis-(S)-MPA ester de... more Comparison of the room-and low-temperature 1 H NMR spectra of the bis-(R)-or bis-(S)-MPA ester derivative of an open chain sec,sec-1,2-diol allows the easy determination of its relative stereochemistry and in some cases absolute configuration. If the diol is anti, its absolute configuration can be directly deduced from the signs of ∆δ T1T2 for substituents R 1 /R 2 , but if the relative stereochemistry of the diol is syn, the assignment of its absolute configuration requires the preparation of two derivatives (both the bis-(R)-and bis-(S)-MPA esters), comparison of their room-temperature 1 H NMR spectra, and calculation of the ∆δ RS signs for the methines HR(R 1 ) and HR(R 2 ) and R 1 /R 2 protons. The reliability of these correlations is validated with 17 diols of known absolute configuration used as model compounds.

Journal of Organic Chemistry, 2005

The absolute configuration of 1,2-, 1,3-, 1,4-, and 1,5-diols formed by two secondary (chiral) hy... more The absolute configuration of 1,2-, 1,3-, 1,4-, and 1,5-diols formed by two secondary (chiral) hydroxy groups can be deduced by comparison of the NMR spectra of the corresponding bis-(R)-and bis-(S)-MPA esters. The correlation between the NMR spectra of the bis-ester derivatives and the absolute stereochemistry of the diol involves the comparison of the chemical shifts of the signals for substituents R 1 /R 2 and for the hydrogens attached to the two chiral centers [H R (R 1 ) and H R (R 2 )] in the bis-(R)-and the bis-(S)-ester and is expressed as ∆δ. RS Theoretical calculations [energy minimization by semiempirical (AM1), ab initio (HF), DFT (B3LYP), and Onsager methods, and aromatic shielding effect calculations] and experimental data (NMR and CD spectroscopy) indicate that in these bis-MPA esters, the experimental ∆δ RS values are the result of the contribution of the shielding/deshielding effects produced by the two MPA units that combine according to the actual stereochemistry of the diol. The reliability of these correlations is demonstrated with a wide range of diols of known absolute configuration derivatized with MPA and 9-AMA as auxiliary reagents. A simple graphical model that allows the simultaneous assignment of the two asymmetric carbons of a 1,n-diol by comparison of the NMR spectra (∆δ RS signs) of its bis-(R)-and bis-(S)-AMAA ester derivatives is presented.

Organic Letters, 2005

The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperatu... more The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperature NMR of a bis-MPA ester derivative. The assignment requires the analysis of just the methylene protons, is not limited by the absence of signals from the R group of the diol, and requires a very small and recoverable sample.

Journal of The American Chemical Society, 2009

Cyclization is a powerful strategy for inducing antiparallel -sheet secondary structure in relati... more Cyclization is a powerful strategy for inducing antiparallel -sheet secondary structure in relatively short peptide segments. Biological examples feature cyclization via the backbone, 1 as seen in gramicidin S and θ-defensin, and cyclization via side chains (disulfide formation), 2 as seen in tachyplesins and protegrins. These natural prototypes have inspired the use of cyclization in -sheet design efforts aimed at both structural and functional goals. For example, both backbone and side chain cyclization have been used to generate peptides that serve as spectroscopic references for the -sheet conformations adopted by flexible, linear peptides. 3 Cyclic -sheet scaffolds have provided a fruitful basis for development of peptides with a variety of biological activities, including antibiotics, vaccine epitopes, RNA ligands, and, perhaps most intriguingly, helixmimetic inhibitors of protein-protein recognition. 4 The demonstrated utility of cyclically enforced antiparallel -sheet scaffolds raises the prospect that analogous parallel -sheet scaffolds would be comparably useful. Biology does not offer a clear basis for achieving this structural goal in relatively small molecules. Interstrand disulfides seem to be incompatible with a parallel -sheet secondary structure, given the rarity of such crosslinks in proteins. 5 -Strand-forming segments must be linked N-terminus-to-C-terminus within peptides and proteins; therefore, covalent connection of -strands in parallel orientation requires a peptidic linker that is at least as long as the -strands themselves. This topological limitation has inspired many efforts to devise small nonpeptide units that can be used to connect peptide segments in a C-to-C or N-to-N fashion. 6-8 Such units should ideally have a strong turn-forming propensity that can encourage -sheet interactions between attached peptide segments. We have developed both preorganized C-to-C linkers and preorganized N-to-N linkers that promote (but do not enforce) parallel -sheet formation between peptide segments in aqueous solution ). 9 Here we evaluate these linkers in the context of backbone cyclization. Unexpectedly, we find that only one of the two turn units needs to be preorganized to enforce a high level of parallel -sheet folding.

Journal of The American Chemical Society, 2008

Short peptides that adopt specific secondary structures in aqueous solution have proven to be inv... more Short peptides that adopt specific secondary structures in aqueous solution have proven to be invaluable tools for elucidating the balance of forces that controls the stability of these common protein substructures. Rules for the design of sequences that fold autonomously into the R-helical conformation have been available for nearly two decades, 1 and the analysis of R-helical folding preferences is now a mature field. Guidelines for the design of antiparallel -sheet-forming sequences have emerged more recently, enabling fundamental studies of the origins of antiparallel -sheet stability. 2 Parallel -sheet is common in proteins, but this structural motif has received little attention to date because the rules necessary to design molecules that will adopt this secondary structure in aqueous solution are not yet fully developed.

Angewandte Chemie-international Edition, 2007

Understanding the factors responsible for the stability of common protein secondary structures ha... more Understanding the factors responsible for the stability of common protein secondary structures has been a longstanding goal. Analysis of b sheets has lagged behind the study of a helices because the development of suitable model systems has been more challenging for the former than for the latter. [1] Over the past decade, however, rules for the design of b sheets that fold autonomously in water have been elucidated, with a particular emphasis on two-stranded antiparallel b sheets ("b hairpins"), which represent a minimum increment of this secondary structure. These model systems have proved useful for evaluating the contributions of factors such as side-chain-side-chain interactions, [4] interstrand linker composition, strand length, and strand number to the stability of b sheets. [7] As an outgrowth of this work, b hairpins have emerged as excellent platforms for the evaluation of noncovalent interactions that do not necessarily occur naturally in b sheets. [8] b Hairpins and hairpin-like molecules stabilized by cyclization are also attractive systems for biomedical applications and fundamental studies. [9] The use of designed peptides to probe the origins of bsheet stability, or to study noncovalent attractions between moieties that become spatially juxtaposed upon folding, requires the ability to determine the extent of b-sheet folding in solution. If only two conformational states are populated, unfolded and b sheet, then determining the population of these two states gives the folding equilibrium constant (K fold ), which provides insight into the stability of the folded state (DG fold = ÀR T lnK fold ). For proteins that adopt defined tertiary structures, conformational stability is often assessed by using heat or a chemical denaturant to disrupt the folded state while monitoring the extent of folding by a conformationally sensitive spectroscopic probe (for example, circular dichroism). This approach is convenient because globular proteins are typically completely folded near room temperature and in the absence of denaturant ("native conditions"), which establishes the spectroscopic signature of the folded state, and because it is often straightforward to identify the spectroscopic signature of a fully unfolded state generated at high temperature or high denaturant concentration. In contrast, most of the autonomously folding b-sheet model systems described to date cannot be driven by changing the conditions to the limiting states. Therefore, identifying the spectroscopic signatures for the fully unfolded and fully folded states of b-sheet model systems has frequently required the preparation and characterization of distinct reference peptides or the implementation of elaborate data analysis techniques. [4][7][8] We have recently developed a new approach for studying the conformational stability of small proteins, [11] and here we describe the extension of this approach to b hairpins. This method involves polypeptide analogues in which one backbone amide group has been replaced by a thioester (namely, thiodepsipeptides). The conformational stability is assessed by monitoring the equilibrium constant for a thiol-thioester exchange reaction that causes the full-length thiodepsipeptide to be reversibly cleaved, which precludes adoption of a native-like fold. The "backbone thioester exchange" (BTE) measurements can be conducted under native conditions, and it is not necessary for the full-length molecule to be completely folded under these conditions. Our previous BTE studies have focused on polypeptides that adopt a discrete tertiary structure. [11] Here we show that the BTE method can be extended to a secondary structure model system ).

Angewandte Chemie, 2007

Understanding the factors responsible for the stability of common protein secondary structures ha... more Understanding the factors responsible for the stability of common protein secondary structures has been a longstanding goal. Analysis of b sheets has lagged behind the study of a helices because the development of suitable model systems has been more challenging for the former than for the latter. [1] Over the past decade, however, rules for the design of b sheets that fold autonomously in water have been elucidated, with a particular emphasis on two-stranded antiparallel b sheets ("b hairpins"), which represent a minimum increment of this secondary structure. These model systems have proved useful for evaluating the contributions of factors such as side-chain-side-chain interactions, [4] interstrand linker composition, strand length, and strand number to the stability of b sheets. [7] As an outgrowth of this work, b hairpins have emerged as excellent platforms for the evaluation of noncovalent interactions that do not necessarily occur naturally in b sheets. [8] b Hairpins and hairpin-like molecules stabilized by cyclization are also attractive systems for biomedical applications and fundamental studies. [9] The use of designed peptides to probe the origins of bsheet stability, or to study noncovalent attractions between moieties that become spatially juxtaposed upon folding, requires the ability to determine the extent of b-sheet folding in solution. If only two conformational states are populated, unfolded and b sheet, then determining the population of these two states gives the folding equilibrium constant (K fold ), which provides insight into the stability of the folded state (DG fold = ÀR T lnK fold ). For proteins that adopt defined tertiary structures, conformational stability is often assessed by using heat or a chemical denaturant to disrupt the folded state while monitoring the extent of folding by a conformationally sensitive spectroscopic probe (for example, circular dichroism). This approach is convenient because globular proteins are typically completely folded near room temperature and in the absence of denaturant ("native conditions"), which establishes the spectroscopic signature of the folded state, and because it is often straightforward to identify the spectroscopic signature of a fully unfolded state generated at high temperature or high denaturant concentration. In contrast, most of the autonomously folding b-sheet model systems described to date cannot be driven by changing the conditions to the limiting states. Therefore, identifying the spectroscopic signatures for the fully unfolded and fully folded states of b-sheet model systems has frequently required the preparation and characterization of distinct reference peptides or the implementation of elaborate data analysis techniques. [4][7][8] We have recently developed a new approach for studying the conformational stability of small proteins, [11] and here we describe the extension of this approach to b hairpins. This method involves polypeptide analogues in which one backbone amide group has been replaced by a thioester (namely, thiodepsipeptides). The conformational stability is assessed by monitoring the equilibrium constant for a thiol-thioester exchange reaction that causes the full-length thiodepsipeptide to be reversibly cleaved, which precludes adoption of a native-like fold. The "backbone thioester exchange" (BTE) measurements can be conducted under native conditions, and it is not necessary for the full-length molecule to be completely folded under these conditions. Our previous BTE studies have focused on polypeptides that adopt a discrete tertiary structure. [11] Here we show that the BTE method can be extended to a secondary structure model system ).

Journal of The American Chemical Society, 2007

Aminoglycosides are clinically relevant antibiotics that participate in a large variety of molecu... more Aminoglycosides are clinically relevant antibiotics that participate in a large variety of molecular recognition processes involving different RNA and protein receptors. The 3-D structures of these policationic oligosaccharides play a key role in RNA binding and therefore determine their biological activity. Herein, we show that the particular NH2/NH3 + /OH distribution within the antibiotic scaffold modulates the oligosaccharide conformation and flexibility. In particular, those polar groups flanking the glycosidic linkages have a significant influence on the antibiotic structure. A careful NMR/theoretical analysis of different natural aminoglycosides, their fragments, and synthetic derivatives proves that both hydrogen bonding and chargecharge repulsive interactions are at the origin of this effect. Current strategies to obtain new aminoglycoside derivatives are mainly focused on the optimization of the direct ligand/receptor contacts. Our results strongly suggest that the particular location of the NH 2/NH3 + /OH groups within the antibiotics can also modulate their RNA binding properties by affecting the conformational preferences and inherent flexibility of these drugs. This fact should also be carefully considered in the design of new antibiotics with improved activity.

Chemical Communications, 2007

A complete characterisation of the protonation equilibrium that accompanies the molecular recogni... more A complete characterisation of the protonation equilibrium that accompanies the molecular recognition of neomycin-B by a specific RNA receptor has been achieved by employing simple NMR measurements.

Chemistry-a European Journal, 2005

The specific interaction of a variety of modified hevein domains to chitooligosaccharides has bee... more The specific interaction of a variety of modified hevein domains to chitooligosaccharides has been studied by NMR spectroscopy in order to assess the importance of aromatic–carbohydrate interactions for the molecular recognition of neutral sugars. These mutant AcAMP2-like peptides, which have 4-fluoro-phenylalanine, tryptophan, or 2-naphthylalanine at the key interacting positions, have been prepared by solid-phase synthesis. Their three-dimensional structures, when bound to the chitin-derived trisaccharide, have been deduced by NMR spectroscopy. By using DYANA and restrained molecular dynamics simulations with the AMBER 5.0 force field, the three-dimensional structures of the protein–sugar complexes have been obtained. The thermodynamic analysis of the interactions that occur upon complex formation have also been carried out. Regarding binding affinity, the obtained data have permitted the deduction that the larger the aromatic group, the higher the association constant and the binding enthalpy. In all cases, entropy opposes binding. In contrast, deactivation of the aromatic rings by attaching fluorine atoms decreases the binding affinity, with a concomitant decrease in enthalpy. The role of the chemical nature of the aromatic ring for establishing sugar contacts has been thus evaluated.

Chemical Communications, 2008

A simple NMR methodology, through the formation of chiral BINOL borates in the NMR tube, and that... more A simple NMR methodology, through the formation of chiral BINOL borates in the NMR tube, and that reunites the advantages of chiral derivatizing (CDAs) and chiral solvating agents (CSAs), is presented for the assignment of the absolute configuration of aand b-hydroxy acids.

Organic Letters, 2006

The absolute configuration of 1,2,3-prim,sec,sec-triols can be assigned by comparison of the 1 H ... more The absolute configuration of 1,2,3-prim,sec,sec-triols can be assigned by comparison of the 1 H NMR spectra of the tris-(R)-and the tris-(S)-MPA ester derivatives. An experimental demonstration of this correlation with 24 triols of known absolute configuration and a protocol using two parameterss∆δ RS (H3) and the difference between ∆δ RS (H2) and ∆δ RS (H3) ) |∆(∆δ RS )|sfor its application to the determination of the absolute configuration of other triols are presented.

Cheminform, 2006

The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperatu... more The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperature NMR of a bis-MPA ester derivative. The assignment requires the analysis of just the methylene protons, is not limited by the absence of signals from the R group of the diol, and requires a very small and recoverable sample.

Chemical Communications, 2005

Chemistry-a European Journal, 2009

The conformational compositions of the tris(α-methoxy-α-phenylacetic acid) ester derivatives of 1... more The conformational compositions of the tris(α-methoxy-α-phenylacetic acid) ester derivatives of 1,2,3-prim,sec,sec-triols are presented. These conformations have been determined by theoretical and experimental data (i.e., energy- and chemical-shift calculations, circular dichroism (CD) experiments, coupling-constant analysis, enantioselective deuteration experiments, and low-temperature NMR spectroscopic studies). A detailed analysis of the anisotropic effects due to the most significant conformers in the 1H NMR spectra supported the correlation between the 1H NMR spectra (ΔδRS value of H(3′) and |Δ(ΔδRS)| parameters) and the absolute configuration of the substrate. The study also allows the identification of the pro-R and pro-S methylene protons from their vicinal coupling constants and relative chemical shifts.

Chemistry-a European Journal, 2005

The absolute configuration of 1,2-diols formed by a primary and a secondary (chiral) hydroxyl gro... more The absolute configuration of 1,2-diols formed by a primary and a secondary (chiral) hydroxyl group can be deduced by comparison of the 1H NMR spectra of the corresponding (R)- and bis-(S)-MPA esters (MPA=methoxyphenylacetic acid). This method involves the use of the chemical shifts of substituents L1/L2 attached to the secondary (chiral) carbon, and of the hydrogen atom linked to the chiral center (CαH) as diagnostic signals. Theoretical (AM1, HF, and B3 LYP calculations) and experimental data (dynamic and low-temperature NMR spectroscopy, studies on deuterated derivatives, constant coupling analysis, circular dichroism (CD) spectra, and NMR studies with a number of diols of known absolute configuration) prove that the signs of the ΔδRS obtained for those signals correlate with the absolute configuration of the diol. A graphical model for the reliable assignment of the absolute configuration of a 1,2-diol by comparison of the NMR spectra of its bis-(R)- and bis-(S)-MPA esters is presented.

Organic Letters, 2010

The absolute configuration of a 1,2-primary/secondary diol can be easily determined by preparatio... more The absolute configuration of a 1,2-primary/secondary diol can be easily determined by preparation of its bis-(R)-and bis-(S)-9-AMA ester derivatives, followed by comparison of the NMR chemical shifts of the diastereotopic methylene protons in the two derivatives. Alternatively, the assignment can be carried out using only one derivative if the evolution with temperature of the signals corresponding to the CrH protons is analyzed.

Journal of Organic Chemistry, 2007

Comparison of the room-and low-temperature 1 H NMR spectra of the bis-(R)-or bis-(S)-MPA ester de... more Comparison of the room-and low-temperature 1 H NMR spectra of the bis-(R)-or bis-(S)-MPA ester derivative of an open chain sec,sec-1,2-diol allows the easy determination of its relative stereochemistry and in some cases absolute configuration. If the diol is anti, its absolute configuration can be directly deduced from the signs of ∆δ T1T2 for substituents R 1 /R 2 , but if the relative stereochemistry of the diol is syn, the assignment of its absolute configuration requires the preparation of two derivatives (both the bis-(R)-and bis-(S)-MPA esters), comparison of their room-temperature 1 H NMR spectra, and calculation of the ∆δ RS signs for the methines HR(R 1 ) and HR(R 2 ) and R 1 /R 2 protons. The reliability of these correlations is validated with 17 diols of known absolute configuration used as model compounds.

Journal of Organic Chemistry, 2005

The absolute configuration of 1,2-, 1,3-, 1,4-, and 1,5-diols formed by two secondary (chiral) hy... more The absolute configuration of 1,2-, 1,3-, 1,4-, and 1,5-diols formed by two secondary (chiral) hydroxy groups can be deduced by comparison of the NMR spectra of the corresponding bis-(R)-and bis-(S)-MPA esters. The correlation between the NMR spectra of the bis-ester derivatives and the absolute stereochemistry of the diol involves the comparison of the chemical shifts of the signals for substituents R 1 /R 2 and for the hydrogens attached to the two chiral centers [H R (R 1 ) and H R (R 2 )] in the bis-(R)-and the bis-(S)-ester and is expressed as ∆δ. RS Theoretical calculations [energy minimization by semiempirical (AM1), ab initio (HF), DFT (B3LYP), and Onsager methods, and aromatic shielding effect calculations] and experimental data (NMR and CD spectroscopy) indicate that in these bis-MPA esters, the experimental ∆δ RS values are the result of the contribution of the shielding/deshielding effects produced by the two MPA units that combine according to the actual stereochemistry of the diol. The reliability of these correlations is demonstrated with a wide range of diols of known absolute configuration derivatized with MPA and 9-AMA as auxiliary reagents. A simple graphical model that allows the simultaneous assignment of the two asymmetric carbons of a 1,n-diol by comparison of the NMR spectra (∆δ RS signs) of its bis-(R)-and bis-(S)-AMAA ester derivatives is presented.

Organic Letters, 2005

The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperatu... more The absolute configuration of 1,2-primary/secondary diols can be easily assigned by low-temperature NMR of a bis-MPA ester derivative. The assignment requires the analysis of just the methylene protons, is not limited by the absence of signals from the R group of the diol, and requires a very small and recoverable sample.