WO2013126370A1 - Hiv-1 protease transition state and uses thereof - Google Patents

Description

HIV-1 PROTEASE TRANSITION STATE AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/601,201, filed February 21, 2012, the contents of which are incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant number GM41916 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to systems and methods for obtaining inhibitors of human immunodeficiency virus- 1 (HIV-1) protease by designing compounds that resemble the charge and geometry of the HIV-1 protease transition state.

BACKGROUND OF THE INVENTION

[0004] Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification before the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.

[0005] HIV-1 protease is an essential enzyme for the human immunodeficiency virus- 1 (HIV-1) viral life cycle and is the target of nine drugs approved by the FDA for the treatment of HIV/AIDS (1). HIV-1 protease inhibitors are administered as part of a drug combination in a treatment termed highly active antiretroviral therapy (HAART), which has become the most effective therapeutic strategy since the discovery of the virus. Nevertheless, mutations in viral enzymes that reduce drug affinity but not catalytic activity continue to breed resistance to HAART. Several mutations in HIV-1 protease have been identified and resistance to all nine FDA approved drugs has been reported (1, 2). Accordingly, attempts to improve inhibitor quality continue with a common goal of increasing the specificity and affinity of enzyme-drug interactions.

[0006] Meek and colleagues published critical insights into the HIV-1 protease reaction mechanism in the early 1990s (3), which contributed substantially to the first generation of clinical HIV-1 protease inhibitors. Subsequent generations of inhibitors relied on crystallographic information and medicinal chemistry to improve binding affinity and oral bioavailability (2). While these approaches have led to many effective HIV inhibitors, none of these drugs are free from the development of resistance. Biologically active mutants of HIV-1 protease recognize and cleave the same precursor polypeptides as their native counterparts; therefore, there remains a common structure-function relationship among them that has not been exploited in existing drugs.

[0007] The present invention address the need for new inhibitors for HIV-1 protease, particularly ones that will be effective against viral strains that have developed resistance to current drugs.

SUMMARY OF THE INVENTION

[0008] The invention provides methods of obtaining inhibitors of human immunodeficiency virus- 1 (HIV-1) protease comprising designing a chemically stable compound that resembles the charge and geometry of the HIV-1 protease transition state.

[0009] The invention also provides systems for obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease comprising one or more data processing apparatus and a computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon that are configured to perform a method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV- 1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.

[0010] The invention further provides methods for screening for a compound that is an inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising the steps of:

(i) determining the molecular electrostatic potential at the van der Waals surface computed from the wave function of a HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state, wherein the HIV-1 protease transition state comprises the structure

(ii) designing a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state,

wherein compound comprises an -NH group and two hydroxyl groups bound to the same carbon atom, and

wherein the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600;

(iii) synthesizing the compound, wherein the compound comprises

two hydroxyl groups bound to the same carbon atom and an -NH group, and three photons with a natural bond orbital charge between +0.500 and +0.600; and

(iv) testing the compound for inhibitory activity to HIV- 1 protease.

BRIEF DESCRIPTION OF THE DRAWINGS

[OOl l] Figure 1. Chemical mechanism of the reaction catalyzed by HIV-1 protease. Structures along the pathway are indicated as follows: (1) enzyme-substrate complex; (2) water attack TS; (3) tetrahedral gem-diol intermediate; (4) proline N-protonation TS; (5) protonated amide intermediate; (6) cleavage of scissile C-N bond TS; and (7) enzyme- product complex. For transition structure 2, the r^c-o) bond distance is defined as the distance between the oxygen of the attacking water and the carbonyl carbon of the peptide. For transition structure 4, the r^.n) is defined as the bond distance between the nitrogen on the proline and the proton on the catalytic aspartate and r(H-o) is defined as the bond distance between the oxygen and proton on the catalytic aspartate. Finally, in transition structure 6, rfC-N) is the bond distance of the scissile bond of the peptide. [0012] Figure 2A-2B. Kinetic isotope effects measured for native and I84V HIV-1 protease. (A) Peptide structure indicating the location of isotopic substitutions used for kinetic isotope effect (KIE) measurements. KIE values are listed for each isotope (values for I84V are in bold). (5) Previously determined crystal structure (17) illustrating the location of the Ile-84 residue that is altered to Val in the drug-resistant mutant.

[0013] Figure 3. Theoretical structures calculated for each step of the HIV-1 protease chemical mechanism using ONIOM(aml :M06-2X/6-31+G**). Calculated structures include: (8) TS for attack of water on the carbonyl; (9) gem-diol intermediate; (10) proton transfer from Asp-25 to proline; (11) protonated amide intermediate; (12) scissile bond cleavage. Distances are shown in Angstroms.

[0014] Figure 4. Theoretical structure for proton transfer from Asp-25 to proline calculated by fixing the r<N-H) bond length at 1.20 A using ONIOM(aml :M06-2X/6-31+G**). Predicted KIEs derived from this transition structure (13) most closely match the experimentally measured values.

[0015] Figure 5A-5C. Electrostatic potential, NBO charges, and inhibitor scaffolds. (A) Structure of the clinical inhibitor indinavir bound to the HIV-1 protease active site obtained from the PDB structure 2AVO (left) and the proton transfer transition structure 13 (right). (5) Electrostatic potential maps extrapolated from single point energy calculations (M06-2X/6-31+G**) of indinavir (left) and 13 (right). NBO charges are indicated in parentheses. (Q Structural interactions with the catalytic aspartates and indinavir (left) and a potential proton transfer TS scaffold (right).

[0016] Figure 6. Synthesis of L-[2-³H]-phenylalanine. ³H is transferred from [1-³H]- glucose to [2-³H]-Phe-OH via a sequence of ³H-transfer reactions. First, ATP-dependent hexokinase catalyzes conversion of [1-³H] -glucose to [l-³H]-glucose-6-phosphate. ³H is then transferred from [l-³H]-glucose-6-phosphate to NAD⁺ by glucose-6-phosphate dehydrogenase (G6PDH), reducing it to [(5)-4-³H]-NADH and forming 6-phosphogluconate. Finally, phenylalanine dehydrogenase (PheDH) is used to produce 2-³H-Phe-OH from [(S)-4- ³H]-NADH and N¾ by reductive amination of phenylpyruvate.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The invention provides a method of obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV-1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease. [0018] The invention also provides a system for obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease comprising one or more data processing apparatus and a computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon that are configured to perform a method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV- 1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.

[0019] The method can include the steps of:

(i) determining the molecular electrostatic potential at the van der Waals surface computed from the wave function of a HIV-1 protease transition state and the geometric atomic volume of the HIV- 1 protease transition state, and

(ii) designing a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.

[0020] As disclosed herein, the HIV-1 protease transition state structure can comprise three protons with natural bond orbital charges of +0.549, +0.564 (diol OHs) and +0.504 ( roline N). The HIV-1 protease transition state structure can comprise

[0021] The method can also comprise synthesizing the putative inhibitor compound and/or testing the compound for inhibitory activity to HIV-1 protease. Preferably, the compound comprises two hydroxyl groups bound to the same carbon atom and an -NH group. Preferably, the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600.

[0022] The invention further provides a method for screening for a compound that is an inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising the steps of:

(i) determining the molecular electrostatic potential at the van der Waals surface computed from the wave function of a HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state, wherein the HIV-1 protease transition state comprises the structure

(ii) designing a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state,

wherein compound comprises an -NH group and two hydroxyl groups bound to the same carbon atom, and

wherein the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600;

(iii) synthesizing the compound, wherein the compound comprises

two hydroxyl groups bound to the same carbon atom and an -NH group, and three photons with a natural bond orbital charge between +0.500 and +0.600; and

(iv) testing the compound for inhibitory activity to HIV- 1 protease. [0023] The HIV-1 protease can be native HIV-1 protease or a mutant form of HIV-1 protease. The mutation can be of the form that causes resistance to currently approved (2012) drugs for treating HIV-1. For example, the mutant form of HIV-1 protease can have valine

(Val or V) substituted for isoleucine (He or I) at amino acid residue 84.

[0024] The invention also provides methods of inhibiting HIV-1 protease comprising obtaining a HIV-1 protease inhibitor by any of the methods disclosed herein or by using the system disclosed herein, and contacting the HIV-1 protease with the compound.

[0025] The invention further provides methods of treating a subject having human immunodeficiency virus- 1 (HIV-1) comprising obtaining a HIV-1 protease inhibitor by any of the methods disclosed herein or by using the system disclosed herein, and administering the compound to the subject in an amount effective to inhibit HIV-1 protease.

[0026] The invention still further provides compounds obtained by any of the methods disclosed herein or by using the system disclosed herein.

[0027] As used herein, a compound resembles the HIV-1 protease transition state molecular electrostatic potential at the van der Waals surface computed from the wave function of the transition state and the geometric atomic volume if that compound has an S_e and S_g >0.5, where S_e and S_g are determined as in Formulas (1) and (2) on page 8831 of Bagdassarian, Schramm and Schwartz, 1996 (55).

[0028] Page 8831 of Bagdassarian et al. 1996 (55) sets forth in part "[a] molecule can be compared to another either geometrically or electrostatically, but ideally, a similarity measure will contain a mixture of both. Consider first the measure

A B

where ■* is the electrostatic potential at surface point i of molecule A, J defines point j of molecule B, and in the numerator is the spatial distance squared between point i on A and j on B. nA and «Β refer to the number of surface points on each molecule. The double summation is therefore over all possible interactions between points on the two molecules, and a is the length scale for the interaction between i and j. The numerator compares A to B for a particular orientation of molecule B relative to molecule A. The denominator serves as a normalization factor for the comparison of A to itself and for B to itself. Here, ^ refers to the distance between i and j on the same molecule. The distance between points is squared to decrease computation time. Consider also a second, purely geometrical measure:

nA f?B

[0029] This invention will be better understood from the Experimental Details that follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

Introduction

[0030] HIV-1 protease is a homodimer of 99 amino acid subunits, each of which contribute a catalytic aspartate to the enzyme's active site (Asp-25 and Asp- 125) (4, 5). The catalytic mechanism and structural details of the HIV-1 protease reaction are illustrated in Fig. 1 (structures 1-7). The cycle initiates with water activation by hydrogen-bonding interactions with the active site aspartate residues (structure 1) (6, 7). Bell-shaped pH-rate profiles (8-12) have revealed an acidic pH optimum and structural studies (13, 14) suggest that the reactant-bound enzyme contains a single protonated Asp (with a di-Asp, net charge of -1) facilitating a general acid-base mechanism, which is common among many Asp proteases (7, 15). Nucleophilic attack by the water generates a reversible gem-diol intermediate, (structure 3) which has been observed structurally (16-18) and identified experimentally (19, 20). A series of kinetic studies have concluded that the breakdown of structure 3 to generate the products (structure 7) is the rate-liming chemical step (9-11, 19, 21), denoted by £¾. Prior ¹⁵N isotope effect studies suggest that N-protonation contributes to ks (22) but little additional experimental evidence distinguishes whether the rate-limiting chemical step is proton transfer to the leaving nitrogen (3 5), breakage of the C-N bond (5 - 7), or a concerted N-protonation and C-N bond breakage (3 -> 7) and thus which transition state (4, 6, or a concerted 4/6) has the highest energy barrier.

[0031] Theoretical studies of the chemical mechanism of HIV-1 protease have included electronic structure calculations (23-26), QM/MM studies (27-29), and molecular dynamic simulations (30-32). Early ab initio calculations by Lee et al. proposed that the irreversible step of the protease reaction comes after the formation of structure 3 (24). Later, Okimoto et al. used HF and a simple model system to calculate that the second step of the reaction consists of a stepwise rate-limiting proton transfer (structure 4) followed by cleavage of the C-N bond (structure 6) (23). A more recent QM/MM molecular dynamics study on the entire enzyme characterized the two reaction barriers and confirmed earlier findings that structure 4 is rate-limiting (21 kcal/mol barrier) (29). Additional insights have stemmed from molecular dynamic simulations, which have shown that Asp- 125 is responsible for the initial protonation of the carbonyl oxygen and that Asp-25 subsequently protonates the amide nitrogen (29, 30). Some general conclusions from these studies are that structure 3 is a stable intermediate in the reaction and that the bystander water, believed to maintain hydrogen bonding within the active site, has a high binding energy (27, 32).

[0032] A powerful technique in determining the chemical details of a reaction mechanism is to use experimentally measured kinetic isotope effects (KIEs) to constrain theoretical predictions (33). This method for elucidating enzymatic transition structures has provided sufficiently robust transition-state knowledge to permit design and synthesis of powerful transition-state mimics (34). Here, transition-state analysis is applied to the study of the chemical mechanism of HIV-1 protease. Experimentally measured intrinsic KIEs are used to evaluate candidate transition-state structures originating from the distinct chemical barriers and intermediates in the reaction cycle of HIV-1 protease. The availability of a geometrically accurate transition structure provides chemical resolution of the reaction and allows development of transition-state inhibitors of the enzyme. Understanding transition- state features for native and resistant enzymes also provides insights into the mechanism of resistance.

Materials and Methods

[0033] Isotopic Labeling and Synthesis of Peptides. Peptide substrates (Fig 2A, Table 1) were synthesized by sequentially coupling FMOC-protected amino acids onto a CLEAR- amide resin (100-200 mesh 0.43 mmol/g) followed by N-acetylation, resin cleavage, precipitation, and purification. Isotopic labels were incorporated at the remote positions by acetylation with either [³H₃]Ac₂0 (purchased) or [l-¹⁴C]Ac₂0 (purchased), and isotopic labels at the scissile positions were incorporated by coupling the appropriate labeled amino acid from the following: FMOC-[l-¹⁴C]Phe-OH (purchased), FMOC-[¹⁵N]Pro-OH (purchased), FMOC-[a-³H]Phe-OH (synthesized), or FMOC-[¹⁸0₂]Phe-OH (synthesized). Peptides were purified by semi-preparative reverse-phase HPLC using a linear gradient (3- 40% acetonitrile in water with 0.1% TFA over 40 min). The purified non-radioactive peptides were characterized by MS-ESI, and radioactive peptides were confirmed by co- elution with the non-radioactive peptide using analytical reverse-phase HPLC over the same linear gradient. FMOC-[l-¹⁴C]Phe-OH, [³H]Ac₂0, and [l-¹⁴C]Ac₂0 were purchased from American Radiolabeled Chemicals, Inc., and FMOC-[¹⁵N]-proline was purchased from Cambridge Isotope Laboratories, Inc.

[0034] Synthesis of Fmoc-[¹⁸ O] 2-Phe-OH. ¹⁸0 incorporation into phenylalanine was accomplished by exchange in acidic H₂ ¹⁸0 (97%, Cambridge Isotope Laboratories, Inc.) at as previously described (45). [¹⁸0₂]Phe-OH was then reacted with FMOC-Osu (dissolved in ice cold acetone) under basic conditions ( aHC03 to H₂ ¹⁸0 (97%) to pH 9). The reaction was monitored by loss of ninhydrin reactivity and TLC and complete conversion to FMOC- [¹⁸0₂]-Phe-OH was confirmed by mass spectrometry.

[0035] Synthesis of Fmoc-[a-³H]-Phe-OH. [a-³H]-Phe-OH was synthesized by reductive amination of phenylpyruvate through a series of enzymatic ³H transfer reactions (Fig. 6). A reaction mixture of 4 mM [l-³H]glucose, 50 mM ATP, 160 μΜ NAD, 250 mM NH₄C1, 10 mM MgCl₂, and 10 mM phenylpyruvate in 50 mM Tris-HCl pH 8.5 was prepared and the following enzymes were added sequentially (0.5 units each): 1) phenylalanine dehydrogenase (Sigma), 2) glucose-6-phosphate dehydrogenase (Sigma), 3) hexokinase (Sigma). The reaction was completed in approximately 45 minutes at room temperature. Purification of the phenylalanine product was accomplished by reverse-phase HPLC over a linear gradient (5-50% acetonitrile in water and 0.1% TFA over 20 minutes) on a CI 8 column (Waters Delta Pak, 300 x 3.9 mm, 15 μιη, 300 A), with the peak corresponding to phenylalanine eluting at approximately 25% acetonitrile. [a ^"3H]Phe-OH was then reacted with FMOC-Osu (dissolved in ice cold acetone) under basic conditions ( aHC03 to H₂0 to pH 9) and conversion to FMOC-[a-³H]-Phe-OH was confirmed by loss of ninhydrin reactivity and TLC analysis. [0036] Protease expression and purification. Both HIV-1 protease (PR) constructs (native and I84V) bearing the five background mutations Q7K, L33I, L63I (for restricted auto-proteolysis), and C67A and C95A (for restricted Cys thiol oxidation) were provided in pET-l la vectors. The constructs were transformed into BL21(DE3) E. Coli cells and expressed and purified from inclusion bodies according to an established protocol (46).

[0037] Kinetic Isotope Effect Measurements. Kinetic isotope effects (KIEs) were measured using the competitive isotopes method (47). The KIE on V/K was determined by the relative change in the ratio of light and heavy peptides (each bearing either ¹⁴C or ³H radiolabels) in the unreacted substrate vs. remaining substrate after multiple reaction cycles. Peptides were radiolabeled with either ³H or ¹⁴C as shown in Table 1. KIEs were measured by mixing the heavy and light peptides such that the cpm ratio of ³H:¹⁴C was 3 : 1 (150,000 cpm:50,000 cpm), with a total peptide concentration kept at 0.5 mM in a 200 μΐ reaction volume. 50 μΐ was taken immediately to determine the ratio of the radiolabels in unreacted peptides, Ro (R = Scissile Bond Heavy Isotope/Scissile Bond Light Isotope). -150 nM protease was added (0.5 μΐ of 1 mg/ml stock) to the remaining 150 μΐ and the reaction was stopped at -70% completion (f = 0.7). The unreacted substrates were purified from products over 0.5 ml AG1-X8 anion exchange columns (equilibrated with 2 column volumes 0.1 N NaOH and 2 column volumes of water), which bind the carboxylate generated in the product peptides (22). The ratios of isotopes of the unreacted substrates at f = 0 (Ro) and f = 0.7 (R_f) were determined by liquid scintillation counting, and the KIEs were determined from Equation 3 (48):

[3] KIE = ln(l - yin[(l -j)(R Ro)l

Each reported KIE results from a minimum of 3 repeats of 6 reactions (n > 18), which were determined from the average of 10 cycles of scintillation counting at 10 minutes per sample.

[0038] Computational methods. The mechanism of HIV-1 protease was studied using the QM/MM two-layer ONIOM (M06-2X/6-31+G**:AMl) method as implemented in Gaussian 03 and 09 (38, 49). A model system of the active site containing 102 atoms was used in the calculations and was derived from the crystal structure of HIV-1 protease co- crystallized with the gem-diol intermediate (17). All starting materials and stable intermediates were located as local minima and frequency calculations performed on the optimized structures contained no imaginary frequencies. Transition structures were located as first order saddle points and contained only one imaginary frequency. Additionally, structures for each chemical step of the protease reaction were explored with geometric constraints (as non-stationary points) to determine the range of possible predicted KIEs for each step of the reaction. Forward and reverse intrinsic reaction coordinate calculations were performed as implemented in G09 from each transition structure to verify that each structure lies on the relevant reaction path.

[0039] The isotope effects for each of the theoretical structures were calculated from conventional transition state theory by the method of Bigeleisen and Mayer (50-52) from the scaled theoretical vibrational frequencies. Tunneling corrections were applied on all atoms using a one-dimensional infinite parabolic barrier model and a truncated infinite parabola model was used for proton transfers (53).

[0040] Natural bond orbital (NBO) analysis and single point energy calculations were carried out on the optimized proton transfer transition structure 13 and the clinical inhibitor indinavir from PDB structure 2AVO (54) in Gaussian 09 (M06-2X/6-31+G**). Electrostatic potential maps were visualized in Gaussview 5.0 (isovalue = 0.04) from density and potential cubes acquired from the single point energy checkpoint files in G09.

[0041] General coupling protocol for isotopic peptides. Six isotopic peptides (Table 1) were synthesized for KIE analysis. The reference (light isotope, remotely radiolabeled) peptides were acetylated with either [¹⁴C]- or [³H]-Ac₂0. The carbonyl carbon, amide nitrogen, a-hydrogen, and carbonyl oxygen corresponding to the scissile peptide bond were isotopically substituted by incorporation of Fmoc-[l-¹⁴C]-Phe-OH, Fmoc-[¹⁵N]-Pro-OH, Fmoc-[2-³H]-Phe-OH, and Fmoc-[¹⁸0]-Phe-OH, respectively. The ¹⁵N and ¹⁸0 peptides were remotely radiolabeled via acetylation with [³H]-Ac₂0.

[0042] Peptides were synthesized on CLEAR-amide resin (100-200 mesh 0.43 mmol/g). Coupling was achieved by reacting each amino acid with: lH-Benzotriazolium 1- [bis(dimethylamino)methylene]-5chloro-,hexafluorophosphate

(l-),3-oxide (HCTU); 6-Chloro-l-Hydroxybenzotriazole (6-Cl-HOBt); and N,N- diisopropylethylamine (DIPEA) in Ν,Ν-Dimethylformamide (DMF) for 0.5-2 hours at room temperature. Fmoc deprotection was accomplished at each step by reacting the growing peptide chain with 30% piperidine in DMF for 20 minutes. Between each coupling and deprotection step, the resin was washed three times sequentially with DMF, isopropanol, and methylene chloride. After the final amino acid coupling and Fmoc deprotection, each peptide was acetylated by reacting the peptidyl-resin with AC₂O and DIPEA for 0.5-3 hours. Amino acids bearing stable isotopes were coupled with 3 equivalents (eq) amino acid, 2.9 eq HCTU, 2.9 eq 6-Cl-HOBt, and 5.8 eq DIPEA. Radiolabeled amino acids were coupled with 1.1 eq amino acid (80-125 μϋϊ), 1.1 eq HCTU, 1.1 eq 6-Cl-HOBt, and 2.2 eq DIPEA. The ¹⁴C- remotely labeled peptide was acetylated with 1.2 eq [¹⁴C]-Ac₂0 (50-250 μθί) and 2.4 eq DIPEA. ³H-remotely labeled peptides were acetylated with 1.5 eq [³H]-Ac₂0 (1-5 mCi) and 3 eq DIPEA. Peptides not requiring a remote label were acetylated with 10 eq AC₂O and 20 eq DIPEA.

[0043] Amino acid side chain deprotection and cleavage of the peptide from the resin was achieved by reacting the peptidyl-resin with 95:2.5:2.5 trifluoroacetic acid (TFA), H₂0, and triisopropylsilane (TIS). The peptides were filtered from the resin, precipitated with ice- cold diethyl ether, and then centrifuged. The precipitates were air dried and purified by semi- preparative reverse-phase HPLC using a linear gradient (3-40% acetonitrile in water with 0.1% TFA over 40 min). The peak corresponding to the desired peptide was collected, frozen, and lyophilized. The purified non-radioactive peptides were confirmed by MS-ESI to be 831 Da, and radioactive peptides were confirmed by co-elution with the non-radioactive peptide using analytical reverse-phase HPLC over the same linear gradient.

[0044] Detailed synthesis of Fmoc-[¹⁸ O] -Phe-OH. Phenylalanine (40 mg), dried with P2O5 overnight, was dissolved in 1.0 ml H₂ ¹⁸0 (97%). Dry HC1 gas - generated by reacting 1 mg NaCl with H2SO4 (drop-wise) - was slowly bubbled into the solution and the vial was sealed with a Teflon-line cap. The exchange solution was placed in an oil bath at 60-65° C for 24 to 72 hours. 10 μΐ aliquots were removed at various time intervals to follow the extent of oxygen exchange by ESI-MS. Final incorporation of ¹⁸0 into Phe-OH was measured to be 93.4% and 6.5% into 170 Da ([¹⁸0₂]-Phe-OH) and 168.01 Da ([¹⁸0, ¹⁶0]-Phe-OH) isotopes, respectively, corresponding to a total ¹⁸0 incorporation of 96% Once completed, the obtained [¹⁸0₂]-Phe-OH was stored with P₂O₅ in a vacuum to keep dry.

[0045] Fmoc protection of [¹⁸0₂]-Phe-OH was carried out by dissolving the amino acid in 1.0 ml H₂ ¹⁸0 (97%), followed by addition of NaHC0₃ to bring the reaction pH to 9.0. Fmoc-Osu (121 mg, 1.5 eq) was dissolved in 1.5 ml cold acetone and added slowly to the amino acid solution while stirring on ice. Reaction progress was followed by TLC (8:3 :0.4 CH₂CI3/CH₂OH/H₂O) analysis using ninhydrin reactivity for detection, and after two hours no [¹⁸0₂]-Phe-OH was detected. Complete conversion to Fmoc-[¹⁸0]-Phe-OH was confirmed by mass spectrometry, corresponding to a molecular weight of 833 Da. Subsequently, the reaction mixture was evaporated, washed twice with benzene, and vacuum dried. [0046] Synthesis of Fmoc-[2-³H]-Phe-OH. [2-³H]-Phe-OH was synthesized by reductive amination of phenylpyruvate through a series of enzymatic ³H transfer reactions (Described in the main text and illustrated in Fig. 6). The final yield of [2-³H]-Phe-OH was determined to be approximately 80 μθί - based on scintillation counting of a small aliquot - corresponding to an estimated chemical yield of 1.0 μιηοΐε. The purified [2-³H]-Phe-OH was vacuum dried and dissolved in 1.0 ml water for subsequent Fmoc protection.

[0047] For Fmoc protection, 2 mg of H-Phe-OH was added to the 1.0 ml [2-³H]-Phe- OH solution. 5.25 mg of aHC03 was then added to bring the reaction pH to 9.0. Fmoc-Osu (3.7 mg, 1.1 eq) in acetone at 5° C was then added to the amino acid mixture and stirred on ice for 1 hour and the solution was allowed to warm to room temperature overnight. The reaction was followed by TLC analysis and ninhydrin reactivity. Once the reaction was complete the solution was evaporated, rinsed twice with benzene, and vacuum dried.

[0048] GAMT-NEDT reaction buffer for KIE measurements. 50 mM glycine, 50 mM sodium acetate, 50 mM Tris-HCl, pH 6.0, 0.2 M NaCl, 1 mM EDTA, 1 mM DTT, 0.1 % Triton X-100 (v/v).

[0049] Scintillation counting for KIE measurements. Scintillation counting was performed using 12 x 20 ml vial racks. 10 ml scintillation fluid was added to each sample and samples were counted sequentially for 10 minutes each over 10 cycles of counting. Each counting cycle included: (1) a water control (blank), (2) a ¹⁴C-remote-labeled substrate peptide (15,000 cpm), (3) unreacted heavy/light mixture (¾)_, (4) -70% reacted heavy/light mixture (Rf), (5)-(n)... replicates of (3) and (4) for n reactions. To correct for overlap of signals between ³H and ¹⁴C channels, the ratio of signal of the ¹⁴C-remote-labeled substrate in channel 1 to channel 2 was determined (Equation 4):

[4] r = channel 1 / channel2.

Corrected values for ³H and ¹⁴C cpm were then obtained from Equations 5 and 6:

[5] cpm ³H = channel 1 - channel2 x r

[6] cpm ¹⁴C = channel2 x (1 + r).

[0050] Tables of calculated isotope effects from the fixed parameter methods. Tables 2-8 give examples (from the many exploratory structures calculated) of the redundancies in the predicted IEs for the mechanism of HIV protease. The IEs were calculated from theoretical structures using ONIOM (M06-2X/6-31+G**:aml) and (B3LYP/6-31G*:aml) methods in Gaussian 09. The bond making/breaking distance for each step of the reaction was varied and the grid of IEs is shown below. IEs with the heading KIE are derived from a structure with one imaginary frequency and those shown under EIE are derived from structures with no negative frequencies. The structures that closely match the experimentally measured KIEs are indicated by *at the top of the column in Tables 2, 4, 5, 7 and 8. Intermediates and transition structures for the protonation of the amide and scissile bond breaking are electronically similar, therefore, a grid of predicted IEs using the fixed parameter method gives several matches to the experimental values. To better explore the system, the enzyme pocket was included in the system and the transition structure was calculated without imposing constraints. Unconstrained structures were used for the current study and IEs are shown in Table 1.

Results and Discussion

[0051] Kinetic Isotope Effects. A family of KIEs was measured at atomic positions surrounding the cleavage site of the heptapeptide substrate Acetyl-Ser-Gln-Asn-Phe*Pro-Val- Val-NH2 (*denotes the scissile bond). The individual isotopic substitutions and measured KIEs are listed in Table 1 and shown Fig. 2A. Additionally, KIEs were measured for the multi-drug resistant variant (I84V), which is illustrated in Fig. 2B. KIEs were determined by comparing ³H/¹⁴C ratios from isotopic peptides and reference peptides bearing remote radiolabels (Table 1). Primary carbonyl ¹⁴C KIEs (^UV/K) of 1.029 ± 0.003 and 1.025 ± 0.005, primary ¹⁵N KIEs (^l5 V/K) of 0.987 ± 0.004 and 0.989 ± 0.003, secondary ¹⁸0 KIEs V/K) of 0.999 ± 0.003 and 0.993 ± 0.003, and secondary ³H KIEs (³ V/K) KIEs of 0.968 ± 0.001 and 0.976 ± 0.001 were observed for the native and I84V enzyme, respectively. Derived from the mechanism in Fig. 1 (assuming that any step up to bond cleavage may be isotope-sensitive), Equation 1 can be used to represent the observed KIE (22, 35), where K_eq3 represents the equilibrium constant for formation of the 3 and A¾ is the rate constant for the formation of 7. The forward commitment (cf) represents a probability of substrate bound in the 1 being catalytically turned over rather than being released from the Michaelis complex:

[1] ^m(V/K) = + _C )/(l + Cf).

[0052] The KIE is measured on V/K and reports on isotope-sensitive steps up to and including the first irreversible step of each catalytic cycle (35, 36). A high probability of 1 being turned over to products presents a virtually irreversible step prior to chemistry which can mask expression of the KIE on the chemical step (the intrinsic KIE); the value that reports on the transition state. The reportedly high K_m for this peptide (37) and the observation that the N KIE value is within the limit of all calculated transition-state models for this reaction suggest that the forward commitment can be considered negligible in the analysis, simplifying the equation of the observed isotope effects to the product of the equilibrium isotope effect (EIE) on formation of 3 and the intrinsic KIE determined by the rate-limiting transition state:

[2] ^IE( //Q =

[0053] Theoretical Structures. One difficulty inherent in using the fixed parameter method in calculating KIEs for a multistep enzymatic reaction is that the different chemical steps are electronically similar at points along the reaction coordinate, resulting in similar KIEs for ¹⁴C and ¹⁵N. For example, a late transition structure of 4 (short r^-u ) is electronically similar to an early transition structure of 6 (short r^c-N)) - consequently, both of the theoretical structures are also similar to intermediate 5. Depending upon the model and method used in the calculations, redundancies exist in the KIE predictions for some of the transition structures for each of the chemical steps involved in the HIV- 1 protease reaction. Therefore, the fixed parameter method of locating a single transition structure that matches all experimental KIEs is not appropriate for studying the HIV-1 protease mechanism. Though it is important to note that a number of proton transfer transition structures obtained from using various methods and models exhibit calculated KIEs that most closely match the experimental values. Tables 2-8 show examples of predicted IEs for several structures with fixed bond distances.

[0054] Theoretical structures used for the final calculation of KIEs include an Ala-Pro dipeptide as the substrate - an alanine residue was used in place of the experimental phenylalanine for simplicity - in addition to several important residues in the active site, including the two catalytic aspartates, the nucleophilic water, as well as the structural water (Fig. 3). Theoretical structures were derived from a published crystal structure of HIV-1 protease co-crystallized with a gem-diol intermediate (17). Calculations were performed using both ONIOM (M06-2X/6-31+G**:aml) and (B3LYP/6-31G*:aml) as implemented in Gaussian 09 (38). Theoretical structures were located as local minima for the starting peptide and structures 3 and 5. For each chemical step of the HIV-1 protease reaction, IEs were calculated for each of the structures based upon the lowest energy conformation of the starting material peptide and an intermediate or transition structure similar to the conformational geometry found in the published crystal structure. [0055] To address the difficulties innately associated with the protease reaction, all theoretical structures for transition states 2, 4, and 6 were located as first order saddle points on the potential energy surface. By explicitly including a portion of the active site, it is assumed that geometry constraints are not required to mimic the active site functionality and that the saddle points will represent the on-enzyme transition structures. The theoretical structures from the ONIOM (M06-2X/6-31+G**:aml) method (Fig. 2) were used for the calculation of KIEs shown in Table 1. Both computational methods give similar results; however, ¹⁵N KIEs derived from B3LYP calculations are smaller than expected for most structures (near unity). This observation is consistent with previous studies in the literature that show B1LYP and B3LYP under-predicts ¹⁵N KIEs (39-41). Furthermore, the M06-2X functional gives more accurate results than B3LYP in bond breaking and forming reactions (42).

[0056] Theoretical structures 8, 10, and 12 represent the unconstrained first order saddle points for transition states 2, 4, and 6, respectively (Fig. 3). Transition structure 8 corresponds to the first step of the reaction and indicates that attack of the catalytic water is late - _(C-o₎ is 1.79 A. From the intermediate gem-diol (9), the second step of the reaction involves proton transfer from the aspartyl residue to the nitrogen on proline and occurs at H) of 1.26 A as shown in 10. The third chemical step involves the breaking of the scissile C-N bond of protonated amide (11) which occurs relatively late (r^c-N) is 2.02 A, as shown in transition structure 12). A concerted transition structure could not be located for direct formation of 7 from intermediate 3. Therefore, the protonation and cleavage of the amide are considered to be stepwise, though we cannot entirely rule out the possibility of a concerted reaction.

[0057] KIE predictions. In accordance with the experimental observation of H2¹⁸0 exchange (19), IEs for 10, 11, and 12 were calculated as the product of the EIE for formation of 9 and the KIE of the transition structure (Equation 2) (22). Predicted ¹⁴C and ¹⁵N KIEs for the attack of water on the peptide from transition structure 8 are both too large, and are poor matches to the measured values; though it is unlikely that 2 is KIE-determining because of the experimental observation supporting the reversibility of this step (19). Similarly, ¹⁴C and ¹⁵N KIE predictions for the breaking of the C-N bond of the amide peptide in 12 were also too large to match the experimental values. Clearly the proton transfer transition structure 10 most closely matches the experimentally measured KIEs, though the ¹⁴C and ¹⁵N KIEs are smaller than predicted by approximately 1%. Additionally, the EIEs for the protonated amide intermediate (11) are a relatively close match to the experimentally measured values. This is unsurprising since structures 10 and 11 are nearly identical with the exception of the r^-H) distance of 1.26 A and 1.12 A, respectively.

[0058] The prediction of KIEs for proton transfer often requires a more extensive study than provided by DFT. However, when the experimental data and computational approach also include heavy atom isotope effects associated with proton transfer, predictive ability is improved. Regardless, the ability of any given method to reliably predict isotope effects to greater accuracy than the 1% shown here is questionable. To further explore the nature of the proton transfer, a simplified variational transition state theory approach was applied by varying the r^__H) and r^-o₎ bond distances of transition structure 10 to locate a structure with KIE predictions that most closely match experimental values. Using this approach transition structure 13 (Fig. 4) was located at r^-H) of 1.20 A and r^-o₎ 1 -32 A, which gave the best match to experimental KIEs, bringing the predictions for ¹⁴C and ¹⁵N within 0.7 and 0.8% respectively (Table 1).

[0059] Experimental KIEs were measured for the carbonyl ¹⁸0 and the phenylalanine a-³H, neither of which are directly involved in bond breaking or forming steps of the mechanism. Hydrogen bonding to the carbonyl oxygen of the peptide is the factor with the largest influence on the ¹⁸0 KIE in the mechanism. Hydrogen bonding networks change as the chemical mechanism progresses; however, each of the oxygens at the reactive center are continuously hydrogen bonded to one of the catalytic aspartates, making each step of the reaction electronically similar, resulting in a null change in the ¹⁸0 KIE after substrate binding. An inverse a-³H KIE is attributed to the breaking of the interaction in the starting material peptide of the lone pair of electrons on oxygen to the σ* orbitals of the adjacent hydrogens upon the binding of the peptide to the enzyme. Predictions for a-³H are inverse at every step of the reaction and depend more upon the conformation of the peptide upon binding than upon the chemistry occurring at the adjacent carbonyl position (Fig. 3; Table 1).

[0060] Proton Transfer. An important aspect of proton transfer transition structure 13 is that the model contains three protons directly associated with the substrate (Figs. 4 & 5). The most stable conformation of the proline is a five membered boat, placing the lone pair on the nitrogen in position to accept the proton from Asp25. Stretching of the scissile C-N amide bond is associated with the imaginary frequency for the transition structure of proton transfer, suggesting that amide bond cleavage is associated with this step. However, little change occurs between 10, 13, and 11 (in order of decreasing Γ_(Ν-Η)), with i-₍-c-N₎ Changing only slightly from 1.51, 1.52, to 1.53 A, respectively. Hydrogen bonding to both oxygens of the gem-diol occurs solely with Asp 125 and is maintained throughout the reaction, consistent with previous dynamic, crystallographic, and neutron diffraction studies (15).

[0061] Despite significant exploration, a concerted transition structure was not located for proton transfer and C-N bond cleavage; though it is plausible that the steps occur concurrently. From an examination of simultaneously varying r^c-N) and r^N-H), a concerted transition structure would have to exist with ^_C-N₎ equal to or less than 1.70 A to be consistent with the measured KIEs. However, structures with ^_C-N₎ less than 2.10 A had no associated imaginary frequency. A match of predicted KIEs to the experimental values from a possible concerted transition structure is unlikely.

[0062] Drug Resistance. For an inhibitor to be effective against biologically relevant variants of HIV-1 protease, binding interactions should be maximized with essential regions of the active site. The observation that the transition structures are nearly identical in the native and I84V enzyme (as indicated by experimental KIE data, Fig. 2A, Table 1) indicates that transition-state interactions should be a focus of inhibitor design. Although many powerful HIV-1 protease inhibitors have been developed, drug resistance continues to arise and attempts to understand mechanisms of drug resistance persist. The variant used in these experiments contains a mutation at an active site He residue (I84V), as illustrated in Fig 2B. This variant has displayed resistance to all nine FDA approved inhibitors (2, 43) and has been reported to cause up to a 32-fold reduction in inhibitor binding affinity (44). The observation that the I84V transition structure is identical to that for the native enzyme indicates that the drug resistant behavior that arises is independent from transition-state interactions. A stable mimic of transition structure 13 shown in Fig. 4 should be an effective inhibitor both the native and I84V HIV-1 protease, and likely an effective inhibitor against all biologically relevant HIV-1 protease variants.

[0063] An electrostatic potential and natural bond orbital (NBO) charge comparison of 13 and a well-characterized clinical inhibitor, indinavir, is shown in Fig. 5. Electrostatic potential maps provide useful blueprints for inhibitor design since they provide shape and charge details of the reactant at the moment of the transition state. The map of indinavir reveals a discrepancy common among the transition structure and the existing FDA approved HIV-1 protease inhibitors. All clinically approved inhibitors posses the gem-diol mimic, but lack the charge character that results from the additional proton present in 13 (Figure 5B and C). The electrostatic maps in Fig. 5B reveal that transition structure 13 has three protons with BO charges of +0.549, +0.564 (diol OH's), and +0.504 (proline N), where the indinavir has one proton within the diol mimic with an NBO charge of +0.514. A transition-state analogue scaffold proposed from the electrostatic potential map in Fig. 5B is shown in Figure 5C (right) with a focus on transition structure features. While the catalytic aspartic acids provide a proton to both the transition-state mimic and indinavir upon inhibitor binding, the crucial feature of the proposed transition-state inhibitor scaffold is the increased protonation state of the nitrogen, and should be a focus of future inhibitor design.

[0064] Conclusions. Using a combination of experimentally measured KIEs and theoretical calculations, the structure of the rate-limiting transition state in the HIV-1 protease reaction has been resolved. The findings confirm previous observations that proton transfer is rate limiting (22, 23, 29). An electrostatic map structure has been established to serve as a blueprint for improved inhibitor design. Inhibitors of HIV-1 protease are usually referred to as transition-state analogues; however, no transition structures have been previously reported from any combination comprehensive KIE analysis and computational approaches as established here. Additionally, a highly drug-resistant mutant form of the HIV-1 protease enzyme has been confirmed to share nearly identical transition-state features with the native enzyme, indicating that drug resistance in this mutant arises from alterations in the enzyme distant from transition-state interactions. Similarity in the transition state features suggests that a robust transition-state mimic would serve as an effective inhibitor of both the native and I84V enzyme, and likely other mutants that share common transition state features.

Table 1. Experimental and Theoretical Kinetic Isotope Effects

Peptide Substrates Experimental KIEs^'¹ Theoretical Structures and KIE predictions

Heavy Light Native I84V 8 9 10 11 12 13

Ac-SQN[ 1 - ¹⁴C]F *P W-NH₂ [³H₃]Ac-SQNF*PVV-NH₂ 1.029(3) 1.025(5) 1.073 1.017 1.020 1.019 1.057 1.025

[³H₃]Ac-SQNF*[¹⁵N]PVV-NH₂ [¹⁴C]Ac-SQNF*PVV-NH₂ 0.987(3) 0.989(3) 1.000 1.002 0.996 0.992 1.015 0.995

[³H₃]Ac-SQN[¹⁸0]F*PVV-NH₂ [¹⁴C]Ac-SQNF*PVV-NH₂ 0.993(3) 0.999(3) 0.991 0.995 0.994 0.994 0.996 0.995

Ac-SQN[a-³H]F*PVV-NH₂ [¹⁴C]Ac-SQNF*PVV-NH₂ 0.968(1) 0.976(1) 0.938 0.922 0.919 0.908 0.874 0.917

*Denotes the location of the scissile bond

^†Experimental errors are shown in parentheses (x 10^~3) following the KIE values and reflect the standard error of the mean resulting from n > 18 independent measurements.

^KIEs obtained using the [³¾]-Ac remote radiolabel are corrected for a ³H effect that was measured to be 0.974(2) and 0.976(2) for the native and I84V enzyme, respectively. Table 2. Transition state for attack of water (M06-2X/6-31+G** full model system)

Table 3. gem-diol intermediate (M06-2X/6-31+G** full model system)

Table 4. Transition state for proton transfer (M06-2X/6-31+G** full model system)

Table 5. Protonated amide intermediate (M06-2X/6-31+G** full model system)

EIE*

[³H₃]Ac-SQNF*[¹⁵N]PVV-NH₂ 0.992

Ac-SQN [1-¹ C] F*P W-NH₂ 1.019

[³H₃]Ac-SQN[¹⁸0]F*PW-NH₂ 0.994

[³H₃]Ac-SQN[¹⁸0]F*PW-NH₂ 0.998

Ac-SQN[a-³H]F*PW-NH₂ 0.908 Table 6. Transition state for scissile bond breakin M06-2X/6-31+G** fu 1 model system)

Table 7. Transition state for proton transfer (B3LYP/6-31+G* full model system)

Table 8. Transition state for scissile bond breaking (B3LYP/6-31+G* full model system)

Table 9. Geometries for structures. These coordinates allow for reproduction of the structures using molecular modeling software.

Structure 8 - Attack of water on the carbonyl

ONIOM (M06-2X/6-31+G**:aml)

m06BBts luncon

E(RAM1) = -1.10909212843

E(RM062X) = -1391.31252779 E(RAM1) = -0.657278136005

Zero-point correction= 0.847231 (Hartree/Particle)

Thermal correction to Energy= 0.904747

Thermal correction to Enthalpy= 0.905691 Thermal correction to Gibbs Free Energy= 0.749790

Sum of electronic and zero-point Energies= -1390.917111

Sum of electronic and thermal Energies= -1390.859595

Sum of electronic and thermal Enthalpies= -1390.858650 Sum of electronic and thermal Free Energies= -1391.014552

E (Thermal) CV S

Cal/Mol Cal/Mol- elvin Cal/Mol-Kelvin Total 567.737 208.677 328.122

C,0,3.2021921446,0.497159343,3.4795558309

C,0,3.7546027942,1.7362993951,2.7928538076

0,0,4.0170691655,2.7752047814,3.433181906

C,0, 1.7055026577,0.4796697228,3.4590629443

C,0, 1.0611322799,0.1603806038,2.096774233

0,0,1.7703070488,-0.0881724735,1.091 1172549

0,0,-0.2057574016,0.1836041289,2.1020075747

N,0,4.0136474368, 1.6353869772,1.4337114938

C,0,4.3041347785,2.7735249585,0.6268044692

C,0,3.1754685588,3.3621417447,-0.232039206

0,0,3.3719379575,4.4867756622,-0.7562510903

N,0,1.9963624833,2.6907066825,-0.4348531816

C,0,0.9933251134,3.1968757004,-1.3226128748

C,0,0.057766165,4.2646630643,-0.728692657

0,0,-1.1820655726,4.14986163,-0.8704891443

N,0,0.6100338353,5.3479412909,-0.0967000781

C,0,-0.1710704506,6.3972928681,0.473093489

C,0,3.1909957407,-0.5911099835,-3.4399256062

0,0,4.0548276887,-1.2392101958,-2.3712947914

0,0,5.1413477156,-1.7814016516,-2.6656333577

0,0,1.8910337877,-1.310191749,-3.6204948238

0,0,0.8429121497,-0.9729939082,-2.5539054686

0,0,0.9421878646,0.122467774,- 1.941519193

0,0,-0.0787680214,-1.8204107212,-2.3955701419

N,0,3.6083097463,-l.171457407,-1.0662750341

0,0,4.2469845384,- 1.8881024247,-0.0098022237

0,0,3.5162555264,-3.1577835631,0.4635167219

0,0,4.1477507921,-4.2240357669,0.6130607104

N,0,2.1721663628,-3.0604957276,0.7516499271

C,0, 1.4323587429,-4.1587044919, 1.2860751996

C,0,0.1940426925,-4.6162070022,0.5018140178

0,0,-0.6378476558,-5.3373387142,1.1090742982

N,0,0.0376968789,-4.2981581036,-0.8181 14968

0,0,-1.1231752386,-4.6763299201,-1.5662304233

0,0,-4.4090024751,3.9310777616,-0.81 14248278

0,0,-4.4232634459,2.479484808,-1.2332711424

0,0,-5.4849013739, 1.886277272,-1.4286282553

N,0,-3.2141688541, 1.8816657317,-1.3319622709

0,0,-3.0516314816,0.4855710342,-1.6685288885

0,0,-2.4356650671,-0.32374254,-0.5162835132 C,0,-2.2957618055,0.3389500166,-2.985411597 0,0,-2.0264271839,-1.5499377626,-0.8231903428 0,0,-0.9383126602,0.6303772011,-0.2889459766 N,0,-3.063897619,-0.2849655208,0.692205596 C,0,-2.9766916144,- 1.43818069, 1.5830768027 C,0,-3.7140035597,-2.6439950831,1.0070069531 0,0,-4.7795938551,-2.5393124648,0.4119835847 N,0,-3.169867048,-3.8448836629,1.3115416362 0,0,-6.3544145166,-0.1167623954,0.5482838725 H,0,1.2975464045,1.441431421,3.802467527 H,0, 1.3355162173,-0.27061 17298,4.17321 6604 H,0,3.601 1020713,-0.4332730065,2.9939154109 H,0,4.6743586015,3.6162261125,1.2823300937 H,0,3.6320533876,0.8355923745,0.96501329 H,0,1.4778366283,3.6627081075,-2.2299774446 H,0,0.3376357137,2.3349644768,-1.6510172647 H,0,1.8466579282,1.7643416954,-0.0810664854 H,0, 1.6017212037,5.4435503244,-0.0689883269 H,0, 1.4477136162,- 1.0190321245,-4.584835511 H,0,2.0267465969,-2.397750907,-3.6566598819 H,0,3.0011080369,0.482473093,-3.1674768145 H,0,5.2786813998,-2.2109331952,-0.3359930828 H,0,2.7324872208,-0.725759238,-0.8686689602 H,0,1.0586694741,-3.8982629277,2.3201451491 H,0,2.1024806993,-5.0657089112, 1.3694434523 H,0, 1.730503633,-2.163168253,0.7350673977 H,0,0.6096326977,-3.5963496536,-1.2381 130894 H,0,-1.9280426634,-1.6808966939,1.7963129561 H,0,-2.2000157272,-3.92347085,1.5869985264 H,0,-4.0600034568,0.0686576559,-1.7718594325 H,0,-1.2512364097,-1.586818991,-1.5076200186 H,0,-0.169020822,0.382331 518,-0.9177046892 H,0,-6.1899163532,0.4684214686,-0.2058000097 H,0,-5.8130343178,-0.9093448056,0.3941448288 H,0,-2.8449690487,0.8908334718,-3.7534076653 H,0,-1.2870095231,0.7555647242,-2.9039116394 H,0,-2.2167356811,-0.7060572444,-3.2887467994 H,0,-5.1504598919,4.4725450147,-1.40032966 H,0,-4.7127956446,3.9735165604,0.2389421051 H,0,-3.4251342727,4.398510904,-0.9161974077 H,0,-3.6011100607,-4.6626138019,0.905845086 C,0,-3.4082395118,0.9547111246,1.4194574845 H,0,-2.6849312344,1.7386683057,l.1817159474 H,0,-4.4242857641 , 1.2709967675, 1.1589577813 C,0,-3.3169423709,0.5369269963,2.8896128524 H,0,-3.9896977118,1.1248894361,3.519992449 H,0,-2.2860356885,0.6538525492,3.2332167168 C,0,-3.6895118231,-0.9440521918,2.8516186563 H,0,-4.7720743847,-1.059266189,2.7217288453 H,0,-3.3681633561,-1.5026367412,3.7343080461 H,0,-1.867585715,-5.1991637327,-0.9089033489 H,0,-0.8203916953,-5.3708288355,-2.3958096871

Η,0,-1.5993194949,-3.7607986858,-2.0148425

Η,0,-1.2626503096,6.1374792783,0.4344770098

Η,Ο,Ο.1294889064,6.5580032027,1.5438212047

Η,0,-0.0072687378,7.3566034996,-0.0898290436

Η,0,4.3323078576,-1.214760281 1,0.8929562887

Η,0,5.1221574386,2.4975653262,-0.1021089806

Η,0,3.7785370715,-0.61 1925587,-4.3963067848

Η,0,3.5682348972,0.5197611 152,4.5409469626

Η,0,-2.3661370682,2.4077833124,-1.1353190722

Η,0,-0.6317967088,0.4147301779,0.6713816273

Structure 9 - gem-diol intermediate

ΟΝΙΟΜ (M06-2X/6-31+G**:aml) m06BBpxferl 15diolint.2

E(RAM1) = -1.14900902578

E(RM062X) = -1391.33450508

E(RAM1) = -0.70732961 1739

Zero-point correction= 0.848601 (Hartree/Particle) Thermal correction to Energy= 0.906855

Thermal correction to Enthalpy= 0.907799

Thermal correction to Gibbs Free Energy= 0.749748

Sum of electronic and zero-point Energies= -1390.927584 Sum of electronic and thermal Energies= -1390.869330 Sum of electronic and thermal Enthalpies= -1390.868385 Sum of electronic and thermal Free Energies= -1391.026436

E (Thermal) CV

KCal/Mol Cal/Mol-Kelvin Cal/Mol-Kelvin

Total 569.060 209.926 332.646

C,0,2.0187893222,3.1660920278,2.6525221658

C,0, 1.6852313853,4.169981 1871, 1.5596292892

0,0, 1.7437827533,5.3977737558,1.785609326

C,0,0.8102225893,2.4225250394,3.1257685202

C,0,0.4643925737,1.2213269133,2.2459522563

0,0, 1.2432822964,0.7484914514,1.4314915807

Ν,Ο, Ι.3382966544,3.6730878236,0.3224909735

C,0, 1.0031732933,4.5274026144,-0.7728889067

C,0,-0.4799024953,4.4745544903,- 1.1823770181

0,0,-1.1694384826,5.519089056,-1.2032810156

N,0,-0.9962643989,3.2637362219,-1.5726091361

C,0,-2.3600933067,3.0963568975,-1.961037246

C,0,-3.3507651694,2.9594048988,-0.7887230052

0,0,-4.0538129545, 1.9297776314,-0.686278317 N,0,-3.4527522544,4.01 15888391,0.0845706882 C,0,3.9509584012,l.8992503289,-2.2303376075 C,0,4.7430495136,1.3236452659,-1.0686810016 0,0,5.9856966372, 1.2119748188,- 1.1225698492 C,0,3.1216342647,0.8470247263,-2.9037635105 C,0,1.9692751684,0.3592407412,-2.0073535104 O,0, 1.2685425667, 1.2412934644,- 1.4362068916 O,0, 1.8115716118,-0.8812326808,- 1.9020246795 N,0,4.033523867,0.9709801308,0.066945139 C,0,4.6269775937,0.2173303947,l.1227021755 C,0,4.2842194063,-1.2809637729,l.1759303309 0,0,5.1738170114,-2.1004486855, 1.5054030644 N,0,2.9933355646,-1.6877888612,0.9503497504 C,0,2.5855064509,-3.0440391172,1.1421552379 C,0,2.802016147,-3.9878183979,-0.0539576433 O,0, 1.8483269031,-4.6784915426,-0.4757239551 N,0,4.0712083166,-4.0853547799,-0.5671950083 C,0,4.3750040075,-4.8381452838,-1.7404923364 C,0,-5.5363380526,-0.4956830156,-2.0308455269 C,0,-4.3716849175,-1.455136243,-1.9632127912 0,0,-4.5405829668,-2.668620972,-2.1226607577 C,0,-2.0240125385,-l .7787027663,-1.364637062 C,0,-1.1418072064,-1.0983558304,-0.277220635 C,0,-1.221277096,-2.0734367578,-2.6298986446 0,0,0.0977296159,-1.7071346555,-0.1813917074 0,0,-1.0610966859,0.2598372956,-0.6021575008 Ν,Ο,- 1.7024242189,-1.1717957543, 1.0981896655 C,0,-1.4109094651,-2.3672008523,1.9224830253 C,0,-1.8871846791,-3.6803040835,1.2825291417 0,0,-3.0227660711,-4.1140399451,1.4753454638 C,0,-2.1960639122,-2.067525605,3.2099989142 N,0,-0.9798343413,-4.3310066904,0.5284234693 0,0,-5.6357284866,-3.2686494662,0.4932796476 H,0,-0.0675862894,3.0774496575,3.1987553813 Η,Ο,Ο.9853986331,2.0322205935,4.139278703 H,0,2.7981732363,2.447362774,2.27671 10444 H,0, 1.2289505254,5.6013399675,-0.5072725809 Η,Ο,Ι.3384032937,2.6834270388,0.1618571697 H,0,-2.6888284405,3.9737202295,-2.589381845 H,0,-2.4453331219,2.146943475,-2.5645079547 H,0,-0.4409556916,2.4364778827,-1.48499966 H,0,-2.8611584812,4.8042302107,-0.0419558786 H,0,2.6726082272,1.2842992087,-3.8070405941 H,0,3.7343443952,-0.0095385517,-3.2082973263 H,0,3.2859685727,2.7267814666,-1.8606404044 H,0,5.7520688367,0.2888391153,1.054447756 H,0,3.0337059533,1.0326694666,0.0482859797 H,0, 1.4768094073,-3.0448723936, 1.352269671 H,0,3.1381012042,-3.4862534505,2.021 161652 H,0,2.3201315584,-1.0675807694,0.5431900189 H,0,3.4788372918,-5.4320066496,-2.0641 152385

H,0,4.7845209108,-3.4950348094,-0.1987378764

H,0,-6.1364295523,-0.6619535099,-l .1300409059

H,0,-1.536672119,-1.5484478418,3.9094473951

H,0,-0.3333752825,-2.4105724584,2.0873045582

H,0,-0.1381569956,-3.857739993,0.2089649389

H,0,-2.4337147325,-2.7170974578,-0.9751380509

H,0,-0.7528334429,-l .1590154231,-3.0048973567

H,0,-0.43300741 16,-2.8058567388,-2.4386213781

H,0,0.7201328459,-1.3939929901,-0.9194674947

H,0,-0.1179376889,0.5514418909,-0.7387130145

H,0,-5.4090186535,-3.2854520316,-0.4494902954

H,0,-4.8549210584,-3.6369589469,0.9331 106667

C,0,-3.1443180195,-0.8972637679, 1.2799521451

H,0,-3.3589105553,0.1299772555,0.9604204875

H,0,-3.7650235755,-1.5890071653,0.6998103848

C,0,-3.3626788818,-l .1441932772,2.7794819063

H,0,-4.3362817894,-1.6124309022,2.9423873464

H,0,-3.3276175555,-0.2044157354,3.3363123762

0,0,-0.7362419058,0.7589176082,2.4968623946

H,0,-1.0303521598,-0.0332320393, 1.850262179

H,0,-6.1489472134,-0.738221027,-2.9003641912

H,0, 1.6119278443,4.2286347009,-1.6758033258

H,0,4.2881189022,0.6482055592,2.1102988269

H,0,5.2247588238,-5.5422836868,-1.5282499878

H,0,-2.5488637104,-2.9907748319,3.6727258285

N,0,-3.16524931 19,-0.9264596281,-1.6736373798

H,0,-3.0660136874,0.0687418439,-1.495213585

H,0,-5.2269462437,0.5528975904,-2.0599703614

H,0,2.4638902462,3.7476222042,3.5041556456

H,0,4.6882942702,2.3363460692,-2.9542632181

C,0,-4.2859589024,3.981201607, 1.2428299014

H,0,-4.8445918043,3.0086050213, 1.296475055

H,0,-3.6648102758,4.0957652572,2.1724503147

H,0,-5.0260569432,4.8261201706, 1.2032511633

Η,Ο,- 1.9036362406,-2.470700361 ,-3.3866254047

H,0,-1.2886805011,-5.1580596636,0.0375486059

H,0,4.6796850443,-4.1556270184,-2.5801537491

Structure 10 - Proton transfer from Asp25 to proline

ONIOM (M06-2X/6-31+G**:aml) onpxferm06BBgp

E(RAM1) = -1.13096461452

E(RM062X) = -1391.32878389

E(RAM1) = -0.684577621041

Zero-point correction= 0.845716 (Hartree/Particle) Thermal correction to Energy= 0.903571

Thermal correction to Enthalpy= 0.904516

Thermal correction to Gibbs Free Energy= 0.747134 Sum of electronic and zero-point Energies= -1390.929455 Sum of electronic and thermal Energies= -1390.871599 Sum of electronic and thermal Enthalpies= -1390.870655 Sum of electronic and thermal Free Energies -1391.028037

E (Thermal) CV

KCal/Mol Cal/Mol-Kelvin Cal/Mol-Kelvin

Total 567.000 208.847 331.238

C,0,2.0901226619,3.0988660876,2.6387860746

C,0, 1.7835566831 ,4.1126460253, 1.5484669804

0,0,1.8807932396,5.3391769979,1.770424724

C,0,0.8657425882,2.3727752294,3.0981865132

C,0,0.4847526532,l.1852803326,2.2026012743

0,0,1.2766605588,0.7244057288,1.3764314564

N,0, 1.4198418722,3.6262192471 ,0.3109863313

C,0,1.1023235801,4.4902672177,-0.7808007992

C,0,-0.3839910491,4.4753854384,- 1.1818555721

0,0,-1.0611311548,5.5276759057,-1.1628255834

N,0,-0.9197741327,3.2855100446,-1.6126516871

C,0,-2.2906715304,3.1510587624,-1.9878364813

C,0,-3.2659275149,2.9761488141,-0.8075968537

0,0,-3.980970057, 1.9509527278,-0.7400878818

N,0,-3.342390491,3.990357994,0.1109431891

C,0,3.9967809079,1.7885260745,-2.254762021

C,0,4.7711332339,1.2037914364,-1.0853786191

0,0,6.0113985691,1.0630515572,-1.1360260156

C,0,3.149653857,0.7487353414,-2.9252245661

C,0,1.9763317844,0.3019729849,-2.0358312132

0,0,1.2954019411,1.2061777195,-1.4794657995

0,0,1.7827233382,-0.9347004401,-1.9227490323

N,0,4.0497471383,0.8734806485,0.0475057917

C,0,4.6241196861,0.1256999818,1.1177606585

C,0,4.2655696098,-1.3690964394,l.1698374029

0,0,5.1536017779,-2.2067410773,1.4544387994

N,0,2.96158359,-1.7544649981,0.9834012241

C,0,2.5442247251 ,-3.1086732697, 1.1649903802

C,0,2.7039021636,-4.0285840941,-0.0589336085

0,0,1.7245713378,-4.6945057646,-0.4629197488

N,0,3.9531003515,-4.1356893853,-0.6163020632

C,0,4.2019141841,-4.857648535,-l.8218129364

C,0,-5.5253340301,-0.3796954438,-2.0892774771

C,0,-4.4115400066,- 1.3904892847,- 1.9514436743

0,0,-4.6400138033,-2.6024140181,-2.0215591009

C,0,-2.0709551945,-1.7829460887,-1.3609793617

C,0,-1.1429211164,-1.0793433372,-0.330207514

C,0,-1.288530024,-2.175291926,-2.6128541667 0,0,0.0731916011,-1.7188939613,-0.2137629957 Ο,Ο,- 1.03780891 13 ,0.2534454096,-0.7067474644 N,0,-1.6990368312,-1.0826701436,1.0709070843 C,0,-1.4294948273,-2.2626628481, 1.9399162688 C,0,-1.9341908963,-3.5822859506, 1.3363360897 0,0,-3.0745993474,-3.9880771668, 1.5556355226 C,0,-2.2062070953,- 1.9050780505,3.2174051851 N,0,-1.0444568308,-4.2721298493,0.5994011041 0,0,-5.6784068145,-3.0558754448,0.6458044709 Η,Ο,Ο.0023872109,3.0455706735,3.1826480594 H,0,1.0334735129,1.9652547598,4.1063434104 H,0,2.8558738407,2.3662563219,2.2618395689 H,0, 1.3559121462,5.5577299609,-0.5140499158 Η,Ο,Ι.3784229682,2.6357340765,0.1570165818 H,0,-2.6165744059,4.0586923326,-2.5731915613 Η,0,-2.3970588493,2.230023645,-2.6302280696 Η,0,-0.3803632948,2.4466486252,-1.5399976313 Η,0,-2.7357366954,4.77614504,0.0142203715 Η,0,2.7212007106, 1.1852528879,-3.8387805296 Η,0,3.7442975565,-0.1262826567,-3.212450833 Η,0,3.3473699049,2.6323474539,-1.894122385 Η,0,5.7504282949,0.1857973909, 1.0629438769 Η,0,3.0519220486,0.9694029416,0.0337746354 Η,0, 1.4456230798,-3.1031929231,1.4198780209 Η,0,3.1255249621,-3.5759961 122,2.0121859339 Η,0,2.2792679209,-1.1109570923,0.6299898848 Η,0,3.2923847006,-5.4456556625,-2.1173927547 Η,0,4.6857700678,-3.5587501435,-0.2640215302 Η,Ο,-6.127549 151,-0.4385995575,-1.1767677699 Η,Ο,-1.5356898277,-1.3739897396,3.8953152192 Η,Ο,-0.3516842031,-2.3115249236,2.0976812378 Η,Ο,-0.1974901061 , -3.8241426198,0.2564677785 Η,Ο,-2.5137863362,-2.6819580063,-0.9183426851 Η,Ο,-0.7807665182,-1.3018678509,-3.0318286708 Η,Ο,-0.5324732835,-2.9311331021,-2.3857464151 Η,0,0.7204500898,-1.4123337812,-0.9453936177 Η,0,-0.0857103551,0.5508929541,-0.7871451846 Η,Ο,-5.4905910503,-3.1123597659,-0.3039217738 Η,Ο,-4.9071762577,-3.4635015016, 1.0670237341 C,0, -3.1415047964,-0.7720312949, 1.2555586313 Η,Ο,-3.331450665,0.24833457,0.9026163986 Η,0,-3.7631934098,-1.4749373352,0.694041582 0,0,-3.3559774477,-0.9722088108,2.7614320261 Η,0,-4.3384983081,-1.416678522,2.9367994228 Η,Ο,-3.2971326755,-0.0179667343,3.2894330135 0,0,-0.702600691 ,0.7367847778,2.4342734443 Η,Ο,-1.1028601427,-0.1482945609,1.6805049958 Η,Ο,-6.1560417317,-0.6561139141,-2.9352754476 Η,0, 1.6995069414,4.1782311682,- 1.6869583042 Η,0,4.2748734282,0.5665040125,2.0971711094 H,0,5.0619145541,-5.5641214236,-1.6667970836 H,0,-2.5760625326,-2.8079005521,3.7058576227

N,0,-3.1787304851,-0.9016760784,-1.7044897204

H,0,-3.0302407353,0.0976918125,-1.597316729

H,0,-5.1600689809,0.6448737426,-2.2016641177

H,0,2.542154634,3.6673882526,3.4951191802

H,0,4.7465776476,2.2049799274,-2.978011307

C,0,-4.1438639527,3.9111856205,1.2896140128

H,0,-4.7114444672,2.9425586105,1.3112488507

H,0,-3.4965370121,3.9738711976,2.2062180516

H,0,-4.875306005,4.7641724555,1.310420914

H,0,-1.9908378082,-2.5785071354,-3.3476135925

H,0,-1.3688311578,-5.1137842168,0.1445910372

H,0,4.4652115072,-4.1538113847,-2.6579002658

Structure 11 - Protonated amide intermediate

ONIOM (M06-2X/6-31+G**:aml)

m06BBprotamideint

E(RAM1) = -1.12461532094

E(RM062X) = -1391.32412580

E(RAM1) = -0.672861955514

Zero-point correction= 0.848969 (Hartree/Particle)

Thermal correction to Energy= 0.906976

Thermal correction to Enthalpy= 0.907920

Thermal correction to Gibbs Free Energy= 0.750712

Sum of electronic and zero-point Energies= -1390.926910 Sum of electronic and thermal Energies= -1390.868903 Sum of electronic and thermal Enthalpies= -1390.867959 Sum of electronic and thermal Free Energies= -1391.025167

E (Thermal) CV S

KCal/Mol Cal/Mol-Kelvin Cal/Mol-Kelvin Total 569.136 209.360 330.873

C,0,2.207306525,3.0776066102,2.6437227331

C,0, 1.9245303285,4.0846002959, 1.542246621

0,0,2.0756059244,5.3106604739,1.7378095719

C,0,0.9658071871,2.3939384376,3.1217366911

C,0,0.5272257914,1.2050708769,2.2439488996

0,0,1.307420437,0.733399232,1.3966581201

Ν,Ο, Ι.5238288252,3.5932463231,0.3169815438

C,0,1.2264337343,4.4537912185,-0.7822014321

C,0,-0.2628223531,4.4824049414,-l.1713237825

0,0,-0.9189833908,5.5466811231,-1.1256067572

N,0,-0.8269965399,3.3132224966,-1.6261277555

C,0,-2.2051956275,3.2155572242,-1.9850752244 C,0,-3.1674355468,3.0257104695,-0.796462424 0,0,-3.904417369,2.0138009988,-0.7512749721 N,0,-3.2092638116,4.01 12723884,0.1537450798 C,0,4.0217721622, 1.670281839,-2.2430690842 C,0,4.7713134662,1.0650418034,-1.0674385376 0,0,6.0063235067,0.8800859947,-1.1154939755 C,0,3.1551494832,0.6528104157,-2.9220124634 C,0, 1.9609419122,0.234406941 ,-2.0486329271 O,0, 1.3112024463, 1.1496949474,-1.4747904556 0,0,1.7153990678,-0.996962418,-1.9689056055 N,0,4.0346085315,0.7645391935,0.0615339246 C,0,4.581788187,0.0227685703,1.1499136944 C,0,4.1957487208,-1.4650801343,1.2057893553 0,0,5.0718217453,-2.3249292885, 1.4597208897 N,0,2.8801038352,-l.8218538325,1.0436645659 C,0,2.4429925502,-3.1707127682,1.2148878633 C,0,2.5659711742,-4.0752476133,-0.0252589002 O,0, 1.5677806806,-4.7191025606,-0.4209616493 N,0,3.8020007335,-4.1958848122,-0.6067014293 C,0,4.0160672369,-4.9044836174,-l.826973796 C,0,-5.5485607922,-0.2383104337,-2.1069120818 C,0,-4.473686168,-1.2875153819,-1.9487773083 0,0,-4.7485273245,-2.4912685027,-1.979938216 C,0,-2.1416398465,-1.7529841804,-1.3781472627 C,0,-1.1727668934,-1.0562309918,-0.384573825 C,0,-1.3859008248,-2.2082572826,-2.6248823109 0,0,0.0176197675,-1.7294194127,-0.253308837 0,0,-1.0369034157,0.2610907301,-0.7751123011 N,0,-1.7299833586,-1.019156436,1.0364780624 C,0,-1.4897327951,-2.1933078982,1.9335971854 C,0,-2.0418552627,-3.501241417,1.3479198351 0,0,-3.1953594859,-3.8604219101,1.5767081466 C,0,-2.2451772576,-1.7857127427,3.2085310399 N,0,-1.1770870537,-4.2327293454,0.6246121 155 0,0,-5.7714501389,-2.8257983586,0.7049147363 H,0,0.1257770515,3.0952472871,3.2111366059 H,0, 1.1332944088, 1.9917535168,4.1322227749 H,0,2.9435591163,2.314989926,2.2677407035 H,0,1.5159154988,5.5157137169,-0.5296307283 H,0, 1.427694214,2.6024668057,0.1868372281 H,0,-2.5207392755,4.1457757646,-2.5398212056 H,0,-2.3386353386,2.3157830855,-2.6518012017 H,0,-0.30933782,2.4607825748,-1.5581771283 H,0,-2.5820997788,4.7832883003,0.0756992322 H,0,2.7475754557,1.098638324,-3.8407250314 H,0,3.7299213726,-0.2371 17476,-3.2037107639 H,0,3.391217616,2.5304500773,-l.8876237722 H,0,5.7094513415,0.0624625031,l. l 111601255 H,0,3.0415796863,0.9078183455,0.0527182253 H,0, 1.3502678587,-3.1546717286, 1.4911063304 H,0,3.0324870479,-3.6608616635,2.0436943799

H,0,2.1966270106,- 1.1491184067,0.7502904926

H,0,3.092869876,-5.4762135061,-2.1117406171

H,0,4.5508533452,-3.6354164886,-0.2610177388

H,0,-6.158993628,-0.263240571,-l.1985659634

H,0,-1.5510202379,-1.2692172519,3.8723627043

H,0,-0.412233183,-2.2634654314,2.0785721592

H,0,-0.309447595,-3.8256150941,0.2815133376

H,0,-2.6111623302,-2.6232238649,-0.9065803072

H,0,-0.8516417935,-1.3664013869,-3.0740350629

H,0,-0.6546897956,-2.9826265061,-2.3796601899

H,0,0.6835391697,-1.4383730225,-0.9865903466

H,0,-0.0737183665,0.5500947175,-0.8274044759

H,0,-5.607716723,-2.9194201633,-0.2463674582

H,0,-5.0209233603,-3.2736825529,l.1222243704

C,0,-3.1637647203,-0.6532659874,1.2358483776

H,0,-3.3207702305,0.3634901986,0.8591507535

H,0,-3.8005688914,-1.3549326916,0.6925414738

C,0,-3.3600339317,-0.8132397178,2.7472589799

H,0,-4.3590477501,-1.2085927309,2.9455273451

H,0,-3.2437957279,0.1484163439,3.250382191

0,0,-0.6471919792,0.7801396932,2.4966405519

H,0,-1.1594309154,-0.2116971939,1.5520503349

H,0,-6.1830812874,-0.5022559199,-2.9540892311

H,0, 1.8067479183,4.1136639554,- 1.6892223506

H,0,4.2230384483,0.4797479782,2.1183517335

H,0,4.869388389,-5.6242780797,-1.6988937554

H,0,-2.6483927688,-2.6681439314,3.7076196593

N,0,-3.2197388355,-0.8397696507,-1.7302864378

H,0,-3.0322596331,0.1558157807,-1.6515103154

H,0,-5.1446413028,0.7703348839,-2.2298384225

H,0,2.6871345674,3.6427032031,3.4868168483

H,0,4.788107213,2.0673629547,-2.9598839403

C,0,-3.9817575147,3.9039069772,1.349964951

H,0,-4.5698106577,2.9473835093, 1.3500545314

H,0,-3.3098477182,3.917576308,2.2508172073

H,0,-4.6933180655,4.7707970907, 1.4208021756

H,0,-2.1089962884,-2.6083732,-3.3406886177

H,0,-1.526058803,-5.0753183475,0.1902695642

H,0,4.2716509381,-4.1925334628,-2.6585476465

Structure 12 - scissile bond cleavage

ONIOM (M06-2X/6-31+G**:aml)

m06BBCNuncon

E(RAM1) = -1.14073599711

E(RM062X) = -1391.32068879

E(RAM1) = -0.696826030057 Zero-point correction= 0.849199 (Hartree/Particle) Thermal correction to Energy= 0.907243

Thermal correction to Enthalpy= 0.908187

Thermal correction to Gibbs Free Energy= 0.750929

Sum of electronic and zero-point Energies= -1390.915400 Sum of electronic and thermal Energies= -1390.857356 Sum of electronic and thermal Enthalpies= -1390.856411 Sum of electronic and thermal Free Energies= -1391.013670

E (Thermal) CV

KCal/Mol Cal/Mol-Kelvin Cal/Mol-Kelvin

Total 569.304 208.825 330.978

C,0,1.0894572547,3. 949561378,2.0737293404

C,0,0.0611495571,4.6998227962,1.2970947095

0,0,-0.4116144514,5.7600840109, 1.7589276164

C,0,0.5372367803,2.6033096692,2.5999207289

C,0,0.3148957671 , 1.5426286166, 1.4909218708

0,0,1.2782345252,0.7869564174,1.2046802351

N,0,-0.2567509665,4.2581204668,0.0222808684

C,0,-1.2918846086,4.8744839152,-0.7453272869

C,0,-2.5273321066,4.0348533985,-1.0986844163

0,0,-3.6124178666,4.626881594,-1.3146128195

N,0,-2.4069078239,2.679 124812,- 1.2934466044

C,0,-3.4948198243, 1.8898051152,-1.7784967648

C,0,-4.4988164115, 1.4173647701,-0.7127581065

0,0,-4.7367265681,0.1945764562,-0.5848775232

N,0,-5.1401841356,2.3742330133,0.0302008169

C,0,3.2821272209,3.3173176881 ,- 1.421590075

C,0,4.3697226977,2.7397767518,-0.5284065338

0,0,5.5637947744,3.0776382653,-0.6757673744

C,0,2.7853559992,2.3082460514,-2.4128612042

C,0, 1.7500778745,1.3505475774,-1.8229829814

0,0,0.5802535072, 1.7027106743,-1.6724920371

0,0,2.2107368813,0.1691051713,-1.5457109819

N,0,3.9688379024, 1.8665352003,0.4567396571

C,0,4.8972402843, 1.1823703365,1.2971949777

C,0,4.9468634911,-0.3451224364,1.1384037328

0,0,6.0554569615,-0.9285734255,1.1813111 104

N,0,3.774246611,-1.0524337405,1.0404012054

C,0,3.76036421 16,-2.4819610525,1.0555646453

C,0,3.9766822102,-3.1601 17567,-0.3096051274

0,0,3.1399488778,-3.9823526894,-0.739703571 1

N,0,5.1357375874,-2.8648016573,-0.9840884319

C,0,5.433055719,-3.3814130145,-2.2795873787

C,0,-5.252653366,-2.9484459356,-0.7502959183

C,0,-3.8489227036,-3.3404973616,-1.1405823619

0,0,-3.4903198878,-4.5222584103,-1.1275236333

C,0,-1.5821675041,-2.6195222374,-1.6259156479 C,0,-0.7304155304,-1.4423860723,-l.173875705

C,0,-1.2373775807,-2.9501647122,-3.0815895629

0,0,0.5230565813,-1.5879920107,-1.1011477848

0,0,-1.2627228674,-0.2706975441,-1.5597972772

N,0,-1.212640952,-1.3258823359,0.7822447591

C,0,-0.2513138672,- 1.9446217506, 1.7213083205

C,0,-0.304069063,-3.4676701426,1.5766799017

0,0,-1.0198654885,-4.1661635671,2.2952776466 C,0,-0.7448689908,- 1.4920960644,3.1050558964 N,0,0.489430707,-3.9879967203,0.6211159039 0,0,-3.8130740753,-4.81 16943017,1.748304631 H,0,-0.4188813845,2.7919434766,3.1052649839 H,0, 1.2550082194,2.1951202485,3.3237457573 H,0,1.9834595926,3.6960435246,1.4238604323 H,0,-1.6680564425,5.7958239848,-0.2098709536 H,0,-0.0048326194,3.31658677 1,-0.21 18777151 H,0,-4.0700491293,2.4730364321,-2.5539065167 H,0,-3.0649784516,0.9562896262,-2.2488378715 H,0,-1.6461241475,2.1773115969,-0.8731111926 H,0,-4.8993131324,3.3320302386,-0.108446943 H,0,2.286138031,2.8409525892,-3.2314991283 H,0,3.6235464666,1.7308458948,-2.8204312895 H,0,2.4328618249,3.7105122047,-0.8002236764 H,0,5.9425576687,1.5653551377,l.1062680183 H,0,3.0025409893, 1.6010509778,0.5129079618 H,0,2.7570880068,-2.8253522989,1.4379257534 H,0,4.5670850146,-2.8607903774,1.7489527767 H,0,2.9071240805,-0.5850306833,0.8577991351 H,0,4.6226411481,-4.0825653601,-2.6147781251 H,0,5.7706423073,-2.2068344364,-0.5879699783 H,0,-5.3507266878,-3.1519901267,0.3212942634 H,0,-0.2473709934,-0.5505241909,3.3506620409 H,0,0.742830237,-l .564294268, 1.4806106671 H,0,0.9712308918,-3.3899482196,-0.046655913 H,0,-1.3399326268,-3.4798493098,-0.998785444 H,0,-1.4695932035,-2.0993242271,-3.7284226434 H,0,-0.1736610344,-3.1859502231,-3.1709550892 H,0,1.4541872253,-0.5089462306,-1.2688856841 H,0,-0.6096949222,0.4670903707,-1.4398576094 H,0,-3.7070899699,-5.0175946402,0.8069601992 H,0,-2.9090144291,-4.6962854755,2.0786783517 C,0,-2.5315689183,-1.4913105495,1.4284356372 H,0,-3.2363151004,-0.7686753972,1.0004533984 H,0,-2.9030178379,-2.5046905191, 1.2452556434 C,0,-2.271610709,-1.285704571,2.9364406503 H,0,-2.8470159135,-2.0066103785,3.5238380576 H,0,-2.5587453813,-0.2763618442,3.2423617455 0,0,-0.8331830555,1.5073288201,0.9720584799 H,0,-1.0071003471,-0.3131629091,0.7988889784 H,0,-5.9588011483,-3.5857594091,-1.2853995337 H,0,-0.8581080758,5.1904617721,-1.7407119457

H,0,4.6291487406, 1.3754531735,2.3776268601

H,0,6.4100001208,-3.9368102451,-2.2559068403

H,0,-0.5089250795,-2.2305674014,3.8743399091

N,0,-2.9968072485,-2.3406295814,-1.4587064985

H,0,-3.2992747798,-1.3727401926,-1.395507774

H,0,-5.4735975695,-1.8943193316,-0.9404914395

H,0, 1.4274494694,4.5400984787,2.9292218099

H,0,3.7358182136,4.1873269015,-1.9690522104

C,0,-6.0313246484,2.0613294279,1.0998648165

H,0,-6.1765995969,0.9504925798, l.1743213873

H,0,-5.620572909,2.4402695241 ,2.075101519

H,0,-7.0276899036,2.5492085834,0.9200756521

H,0,-1.8290702032,-3.814612528,-3.3938403453

H,0,0.4461852969,-4.9832600134,0.456027701

H,0,5.5198047104,-2.5435812254,-3.0239486904

Structure 13 - Proton transfer from Asp25 to proline with Y J-H) bond length at 1.20 A ONIOM (M06-2X/6-31+G**:aml)

E(RAM1) = -1.12705151579 E(RM062X) = -1391.32678196 E(RAM1) = -0.678445029838

Zero-point correction= 0.846194 (Hartree/Particle)

Thermal correction to Energy= 0.903883

Thermal correction to Enthalpy= 0.904827

Thermal correction to Gibbs Free Energy= 0.748185

Sum of electronic and zero-point Energies= -1390.929194

Sum of electronic and thermal Energies= -1390.871506

Sum of electronic and thermal Enthalpies= -1390.870561

Sum of electronic and thermal Free Energies -1391.027204

E (Thermal) CV S

KCal/Mol Cal/Mol-Kelvin Cal/Mol-Kelvin

Total 567.195 208.493 329.682

C,0,2.0132479579,3.1483183533,2.666848309

C,0, 1.6882781068,4.15478821 16, 1.5755892615

0,0, 1.7639784893,5.3836304787,1.7945391083

C,0,0.8047890524,2.3941946553,3.1230563595

C,0,0.446796619, l.1971963217,2.2262405586

0,0, 1.2537093363,0.7669274994,1.3912798313

N,0, 1.3335147554,3.6605644704,0.3382383947

C,0,0.9998415276,4.5180318885,-0.7535099586

C,0,-0.4861821586,4.4750147163,-l .1534579185

0,0,-1.1895326549,5.5093458498,-1.1165059898

N,0,-0.9934747247,3.2796854244,-1.6049859165 C,0,-2.3623310555,3.1186694972,- 1.9769403895

C,0,-3.3270509555,2.8956157586,-0.7961280391

0,0,-4.0163838256,1.8514835658,-0.7470732212

N,0,-3.4242333483,3.8881317476,0.143421711

C,0,3.9587381758, 1.8879369165,-2.21 16463827

0,0,4.7374823648,1.314666406,-1.0393559357

0,0,5.9806362028,1.1979104014,-1.0839128148

C,0,3.1409321698,0.8319854134,-2.8929735281

C,0,1.9709200206,0.3559002309,-2.0150413265

O,0, 1.2606988094, 1.2416413856,- 1.4666537442

O,0, 1.8073425652,-0.8858999448,- 1.9040399465

N,0,4.0155771617,0.9657306228,0.086706773

C,0,4.5978845562,0.2303100553,l.161 1573451

C,0,4.2744753303 ,- 1.2727565724, 1.2032029234

0,0,5.1820710858,-2.0930271918, 1.4764800109

N,0,2.9787805339,-1.6855609177, 1.0172738354

C,0,2.5934374109,-3.0507658481, 1.185022561

C,0,2.7714115705,-3.9536746865,-0.0492391082

O,0, 1.8064089129,-4.638377108,-0.4572547636

N,0,4.020824757,-4.0267708747,-0.6108497203

C,0,4.2821318794,-4.7298382385,-1.8249631264

C,0,-5.5070946657,-0.48748829,-2.1349419626

C,0,-4.3771245972,- 1.4782966077,- 1.9844707276

0,0,-4.5835778283,-2.6943884196,-2.0495911042

C,0,-2.0329120616,-1.8265533353,-1.3807000456

C,0,- 1.1208490497,- 1.1006269191 ,-0.3526131818

C,0,-1.2355448639,-2.2100171994,-2.6258874106

0,0,0.1040711559,-1.7153391539,-0.2231166927

0,0,-1.0446914046,0.2312897687,-0.7259621682

N,0,-1.6896749856,-l.11413321 16, 1.0520020016

C,0,-1.4009784215,-2.2882964703, 1.9279348862

C,0,-1.876762143,-3.6162294139, 1.3198586404

0,0,-3.0106054076,-4.0424909374,1.5332107008

0,0,-2.1916182718,-1.9429678981,3.2001135592

N,0,-0.9694904002,-4.2884463872,0.5894515237

0,0,-5.6251061574,-3.1532646663,0.613442012

H,0,-0.0732263232,3.0477556702,3.20854923

H,0,0.9808948844, 1.9894749635,4.1310103424

H,0,2.7947929474,2.4320887966,2.2908118308

H,0, 1.233036847,5.59020962,-0.4869430275

H,0,1.3026981076,2.6687093661,0.1884122957

H,0,-2.7131992296,4.0296918756,-2.5422281015

H,0,-2.4492107103,2.2087768179,-2.6376669836

H,0,-0.4358406121,2.4523696576,-1.5393814929

H,0,-2.8352910591,4.6891433956,0.0618671379

H,0,2.7105262637, 1.2619428002,-3.8086390719

H,0,3.7577502094,-0.028366414,-3.1776190421

H,0,3.2875635101,2.7150128293,-1.8521289541

H,0,5.7228063344,0.317439024,1.117453899

H,0,3.016239744, 1.0469522461 ,0.0703151081 Η,0, 1.4958458629,-3.0746414332, 1.4423276131

H,0,3.1874206631,-3.5140644409,2.0257110581

H,0,2.2786537319,-1.0505068961,0.6831128534

H,0,3.3856761397,-5.3359436778,-2.1238351363

H,0,4.7408621117,-3.4355948518,-0.2557529384

H,0,-6.1175970764,-0.5557013202,-1.2286779124

H,0,-1.5346617257,-1.3983429552,3.8799279838

H,0,-0.3232021808,-2.3112994897,2.0891137979

H,0,-0.1262319427,-3.8268214903 ,0.2551922993

H,0,-2.4608862264,-2.7315494807,-0.9356820007

H,0,-0.7411207039,-1.3292073071,-3.045426758

H,0,-0.4673001912,-2.9505727971,-2.3895814234

H,0,0.7575177836,-1.3874961227,-0.9447756009

H,0,-0.0984221726,0.5573608084,-0.7910233336

H,0,-5.4335172971,-3.2109043856,-0.3354689502

H,0,-4.8503053483,-3.5496013324,1.0386968284

C,0,-3.1405956251,-0.8294565671, 1.2323022588

H,0,-3.3471010351,0.1857367339,0.8746249112

H,0,-3.740534298,-1.5481507909,0.6681417134

C,0,-3.3551324068,-1.0303011073,2.7375054105

H,0,-4.3310378711,-1.4901271306,2.9102625553

H,0,-3.3113494918,-0.0739393651,3.26261161

0,0,-0.7185451804,0.7129291089,2.4595663603

H,0,-1.1360890044,-0.220932767,1.631428897

H,0,-6.1240379753,-0.7758900685,-2.9870351104

H,0, 1.6030478579,4.2172155125,- 1.6594601809

H,0,4.2276697346,0.6595399904,2.137953913

H,0,5.1590318707,-5.417438827,-1.6801825437

H,0,-2.5484414888,-2.8525014661,3.6856755381

N,0,-3.1544387683,-0.9667479174,-1.7326190862

H,0,-3.0240087177,0.0356255493,-1.6297738665

H,0,-5.1585146713,0.5431110236,-2.2448335348

H,0,2.4515185857,3.726761568,3.5236348149

H,0,4.7040445324,2.3246751296,-2.9275044727

C,0,-4.2110074579,3.7610766142,1.3280300157

H,0,-4.7535600217,2.7779999518, 1.3325407791

H,0,-3.5559034773,3.8189392544,2.2394179905

H,0,-4.9635304041 ,4.5943812934, 1.3754624244

H,0,-1.9255446637,-2.6293654214,-3.3631400275

H,0,-1.2725221067,-5.1386812099,0.135925779

H,0,4.5258272163,-4.0111209149,-2.6542938817

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Claims

What is claimed is:

1. A method of obtaining a putative inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV-1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.

2. A system for obtaining a putative inhibitor of a human immunodeficiency virus-1 (HIV-1) protease comprising one or more data processing apparatus and a computer-readable medium coupled to the one or more data processing apparatus having instructions stored thereon that are configured to perform a method comprising designing a chemically stable compound that resembles the charge and geometry of the HIV-1 protease transition state, wherein the compound is a putative inhibitor of HIV-1 protease.

3. The method of Claim 1 or the system of Claim 2, the method comprising the steps of:

(i) determining the molecular electrostatic potential at the van der Waals surface computed from the wave function of a HIV-1 protease transition state and the geometric atomic volume of the HIV- 1 protease transition state, and

(ii) designing a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state, wherein the compound is a putative inhibitor of HIV- 1 protease.

4. The method of Claim 1 or 3 or the system of Claim 2 or 3, wherein the HIV-1 protease transition state structure comprises three protons with natural bond orbital charges of +0.549, +0.564 (diol OHs) and +0.504 (proline N).

5. The method of Claim 1, 3 or 4, or the system of any of Claims 2-4, wherein the HIV- 1 protease transition state structure comprises

6. The method of any of Claims 1, or 3-5 further comprising synthesizing the compound.

7. The method of Claim 6, wherein compound comprises two hydroxyl groups bound to the same carbon atom and an -NH group.

8. The method of Claim 6 or 7, wherein the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600.

9. The method of any of Claims 1 or 3-8 further comprising testing the compound for inhibitory activity to HIV-1 protease.

10. A method for screening for a compound that is an inhibitor of a human immunodeficiency virus- 1 (HIV-1) protease, the method comprising the steps of:

(i) determining the molecular electrostatic potential at the van der Waals surface computed from the wave function of a HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state, wherein the HIV-1 protease transition state comprises the structure

(ii) designing a chemically stable compound that resembles the molecular electrostatic potential at the van der Waals surface computed from the wave function of the HIV-1 protease transition state and the geometric atomic volume of the HIV-1 protease transition state,

wherein compound comprises an -NH group and two hydroxyl groups bound to the same carbon atom, and

wherein the compound comprises three photons with a natural bond orbital charge between +0.500 and +0.600;

(iii) synthesizing the compound, wherein the compound comprises

two hydroxyl groups bound to the same carbon atom and an -NH group, and three photons with a natural bond orbital charge between +0.500 and +0.600; and

(iv) testing the compound for inhibitory activity to HIV- 1 protease.

1 1. The method of any of Claims 1 or 3-10, or the system of any of Claims 2-5, wherein the HIV-1 protease is native HIV-1 protease.

12. The method of any of Claims 1 or 3-10, or the system of any of Claims 2-5, wherein the HIV-1 protease is a mutant form of HIV-1 protease.

13. The method or system of Claim 12, wherein the mutant form of HIV-1 protease has valine substituted for isoleucine at amino acid residue 84.

14. A method of inhibiting a HIV-1 protease comprising obtaining a HIV-1 protease inhibitor by the method of any of Claims 1 or 3-13, or by using the system of any of Claims 2-5 or 1 1-13, and contacting the HIV-1 protease with the compound.

15. A method of treating a subject having human immunodeficiency virus- 1 (HIV-1) comprising obtaining a HIV-1 protease inhibitor by the method of any of Claims 1 or 3-13, or by using the system of any of Claims 2-5 or 1 1-13, and administering the compound to the subject in an amount effective to inhibit HIV-1 protease.

15. A compound obtained by the method of any of Claims 1 or 3-13, or by using the system of any of Claims 2-5 or 1 1-13.