Kevin DeSisto’s entire Synthesis, Characterization, and Analysis of Proline Templacted Amino Acids Dissertation is presented on these Thesis pages as a viewable and downloadable PDF.

View and Download (or Print) here> 

The first ten pages of the Thesis Chapter 3 are also presented as web text below with accompanying illustrations. The Part 1 and Part 3 sections also have the first ten pages (with illustrations) of the Thesis Chapters 1 and 5.

Kevin DeSisto’s entire Synthesis, Characterization, and Analysis of Proline Templacted Amino Acids Dissertation is presented on these Thesis pages as a viewable and downloadable PDF.

View and Download (or Print) here>   

The first ten pages of the Thesis Chapter 3 are also presented as web text below with accompanying illustrations. The Part 1 and Part 3 sections also have the first ten pages (with illustrations) of the Thesis Chapters 1 and 5.


Section 3.1 – Relevance

Most protein-protein mediated interactions are governed with strict conformational requirements in in well-defined binding clefts. A logical approach to designing competitive ligands would be to build molecules that have access to similar geometric constraints. Furthermore, competitive binding studies(29) have revealed many enzymes bind ligands with preferred and/or required side chain orientations (Figure 3.1). Since proline and PTAAs contain a constrained 5-member pyrrolidine ring, it is of interest to determine the most populated confirmation(s) that the ring adopts.

Based on any one (1) ring confirmation for a given substituted proline, c1 may be extrapolated from the torsion and dihedral angles. In solution, or as a crystal, multiple conformations have been observed for various substituted 5-member rings.(30) It is reasonable to expect our 3-substituted proline analogs may adopt more than one conformation.

Endocyclic torsion angles are convenient to describe a particular conformation for proline or other 5-membered rings. We use computational analysis to calculate and/or verify the conformation(s) for our 3-substituted proline analogs. Experimental methods were developed to gather NMR data (i.e. J-values) and then used to “construct” a model of the conformations(s), which exist in solution. These “models” are compared to optimized computer models and a modified Karplus equation is used to convert the J-values into torsional angles as described by pseudorotational theory.(31)

Section 3.2 –Pseudorotation

3.2.1 – Introduction

Five-membered rings have long been studied(32) with interest regarding their conformational flexibility confined about the exocyclic bonds. Once thought to be flat, 5-membered rings are quite the opposite. Virtually all 5-membered rings, cyclopentyl- as well as heteroatom containing moieties, have a “pucker” with atoms above and/or below the plane. The concept of Pseudorotation was developed by Kilpatrick et al(33) and used to describe the type of “pucker” and its amplitude for various 5-member rings.

The pseudorotational wheel (Figure 3.2) shows a cyclical relationship between all possible ring conformations a 5-member ring may adopt. The wheel is divided into two hemispheres, a Northern (N or ‘N’-type) and a Southern (S or ‘S’-type), each having equal amplitudes and opposite puckers. The phase angle (P) is a point on the pseudorotational wheel (in degrees, o) that describes the ring geometry (i.e. torsional angles). There are two types of pseudorotational conformations general to a 5-member ring; the twist (T) and the envelope (E). An “envelope” consists of one atom above or below the ring’s plane. The superscript and subscript proceeding the conformational assignment refers to the atom(s) endo (above plane) and exo (below plane) for a particular P and its pucker. The amplitude of pucker (fmax) is also a pseudorotational parameter describing the maximum out-of-plane bond angle for each system.

The vast majority of 5-membered rings prefer to populate within a small window of the pseudorotational wheel. N-type conformations generally populate P ~ 0-12owhile S-type conformations generally populate P ~ 174-180o.(34)For most substituted 5-member ring systems, an equilibrium exists between N-type and S-type conformations, however, this is not the case with substituted 5-member rings. Stereoelectronic effects (i.e. gauche effect, anomeric effect, etc.), the geometry of the substituent govern Pand the equilibrium between a pseudorotational pair. A low energy difference between the two conformations in equilibrium would give access to two (2) c1 angles. It is observed on an “NMR time scale” a rapid interconversion and as a result the data are “averaged” coupling constants, if in fact more than one P exists in solution.

Section 3.3 – Nuclear Magnetic Resonance

3.3.1 – Determination of Endocyclic J-values

Our goal is to unambiguously determine the conformation(s) of the 3-substituted PTAAs synthesized (norleucine, Chapter 2). Other than a crystal structure, J-values are the most important experimental data for elucidating the torsional dihedral angles of a 5-membered ring. The norleucine PTAA is used as the model 3-substituted PTAA for the initial conformational studies. The n-propyl side chain is not sterically hindered compared with other aliphatic side chains incorporated into PTAAs and the side chain is covalent, which will not lend itself to stereoelectronic interactions.

Scheme 3.1

In order to simplify the 1H NMR spectrum of the PTAA, the Boc protecting group is replaced with a pivaloyl (Piv) or acetyl (Ac) group analogous to the procedure used in Chaoter 2 (Scheme 3.1). The Piv group, as discussed, does not allow rotation about the amide bond removing any rotomer NMR signals. The Ac group is small enough to leave the ring unbiased toward any stearic interactions between the bulky Piv group and Hd1-2. Once the Piv (Ac) protected PTAA is characterized completely. Several deuterated solvents were examined for use in the experiment. The complexity of the spectrum, due to overlapping signals in the 1-2 ppm region, in d-chloroform made it necessary to find an alternative solvent. Solvent of choice for the NMR experiment was determined by acquiring data using each of the solvents; d8-THF, d3-acetonitrile, and d6-benzene. Deuterated benzene gave the least convoluted spectrum and the proton assignments were made using 2-D COrrelation SpectroscopY (COSY) NMR.

3.3.2 – Spin-spin coupling constants via NMR experiment

The N-Ac-trans-3-n-propylproline benzyl ester 3.2 is our model 3-substituted PTAA for conformational analysis. As described above, d6-benzene was selected as the solvent and the spectrum gave only one unambiguous J-value. The a-proton (Figure 3.4) couples exclusively with the vicinal b-proton resulting in a sharp doublet (d = 4.45 ppm, J = 5.73 Hz).

The other five (5) protons (b, g1, g2, d1, d2) have multiple coupling constants and require further treatment to deconvolute the J-values. The method of deconvolution requires computer simulation(35) of the spectrum and the aid of computer models. The experimentally determined coupling constants (Figure 3.5) are “best” approximations determined from gNMR simulation (see 3.3.3) and used as initial values for PSEUROT calculations. Experimental spin-spin coupling constants are found by determining the distance, in Hz, each signal is split for each proton in the 1H NMR spectrum.

3.3.3 – J-values via NMR simulation (gNMR)

A useful procedure to extrapolate spin-spin coupling constants (J-values) is computer simulation of a NMR spectrum. Commonly used in dynamic NMR studies(36) involving low temperature “freezing” of lower energy minima. We found the signal representing Hb was overlapping with other proton signals from aliphatic side chain.

The simulation program, gNMR (Cherwell Scientific), is straightforward to use. A ‘grid’ of coupling constants is built with experimental values from an actual 1H-proton NMR spectrum. Some coupling constants are clear and unambiguous, however, others may be quite complex due to overlapping signals.  This may be further complicated if the proton is coupled to other protons. Our PTAA of study had complexity arising from both, especially the Hb also being coupled to exo-protons on the n-propyl side chain (Figure 3.4). With the aid of the Ha-b coupling constant, which is a sharp doublet without overlap, the J-values are deconvoluted.

Once the J-value grid is complete (similar to grid above), the program generates an identical spectrum for the given field (i.e. 500 MHz). Careful comparison, on-screen or printed, between the computer generated spectrum (simulated spectrum) and the experimental spectrum reveals the spectra are identical. When some and/or all of the peaks do not match exactly, adjustment of the J-value grid is performed until all signals of the simulated spectrum match with all the signals of the experimental spectrum (see Figure 3.5a). Once the spectrum is simulated to satisfaction, the J-values are used to calculate P, fmax, and the N/S equilibrium using a modified Karplus equation (PSEUROT).

Section 3.4 – Graphical Computation – Molecular Mechanics

Utilizing classical physics and “ball and spring” models, molecular mechanics (MM) is a convenient entry point into conformational description of a molecule or system:

E(steric) = E(t) + E(q) + E(Ǿ) + E(d) (3.1)

The generalized equation(37) allows the calculation of steric energies € due to mechanical deformations where E(steric) is total strain; E(t) is torsional strain; E(q) is bond angle strain, E(Ǿ is torsional strain (dihedral angle), E(d)  is the strain of van der Waals interactions.

Although the equations solved are “easy” relative to a computer (i.e. time required for calculations), all MM fields do not account for quantum mechanics and therefore the results may be interpretative. Also, all computations are based on an abbreviated model of a methyl (-CH3) 3-subsitituted proline rather than the actual n-propyl (-CH2CH2CH3) substituent, which has many degrees of freedom. The N-acetyl moiety is used rather than the N-pivaloyl for complexity issues, as well. – Global Minima via MM

Since 5-membered rings of carbon, with or without heteroatoms may exist in any number of infinite conformations (i.e. the pseudorotational wheel). For the system, lowest energy conformations are expected to populate accordingly. The program PCModel v7.0(Serena Software) had global search capabilities using the gMMX feature that “searches” by structure input followed by calculation of strain energies. The goal is to minimize the total strain of the system by systematic variation of atom locations. The process is repeated until improvement is minimal (below some threshold) then stores and arranges the conformations by energy, the lowest being the global minimum (minima). MMX equations are employed to rapidly calculate the energy (MME) for each conformation. The first 100 structures of lowest energy were compared and conformational rotamers along with duplicate structures were excluded. Two (2) unique ring conformations were discovered among the lowest in energy. Pseudorotation describes one to be a 4-exo envelop (P = 19.8 o, N-type) and other a 4-endo twist (P = 170 o, S-type). The two ring conformers are quite close in energy (MME<0.03 kcal/mol) and are employed as base models for the global minima, which is further minimized using ab initio calculations.