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Single Conformation Spectroscopy of Suberoylanilide Hydroxamic Acid (SAHA): A Molecule Bites its Tail Di Zhang, Karl N. Blodgett, Xiao Zhu, and Timothy S. Zwier J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b12464 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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The Journal of Physical Chemistry

Single Conformation Spectroscopy of Suberoylanilide Hydroxamic Acid (SAHA): A Molecule Bites its Tail Di Zhang, 1 Karl N. Blodgett, 1 Xiao Zhu2 and Timothy S. Zwier1*

1Department

of Chemistry, Purdue University, West Lafayette, IN 47907-2084 U.S.A.

2Rosen

Center for Advanced Computing (RCAC), Purdue University, West Lafayette, IN 47907-2084 U.S.A.

Abstract

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that causes growth arrest and differentiation of many tumor types and is an approved drug for the treatment of cancer. The chemical structure of SAHA consists of formanilide ‘head’ and a hydroxamic acid ‘tail’ separated by an n-hexyl chain, C6H5NH(C=O)-(CH2)6(C=O)NHOH. The alkyl chain’s preference for extended structures is in competition with tail-to-head (T-H) or head-to-tail (H-T) hydrogen bonds between the amide and hydroxamic acid groups. Laser desorption was used to bring SAHA into the gas phase 1

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and cool it in a supersonic expansion before interrogation with mass-resolved resonant two-photon ionization (R2PI) spectroscopy. Single conformation UV spectra in the S0S1 region and infrared spectra in the hydride stretch and mid-IR regions were recorded using IR-UV hole-burning and resonant ion-dip infrared (RIDIR) spectroscopy, respectively. Three conformers of SAHA were distinguished and spectroscopically characterized. Comparison of the experimental IR spectra with the predictions of density functional theory calculations (DFT B3LYP D3BJ/6-31+G(d)) leads to assignments for the three conformers, all of which possess tightly folded alkyl chains that enable formation of a T-H (conformer A) or H-T (conformers B, C) hydrogen bonds.

A modified version of the generalized Amber force field (GAFF) was

developed to more accurately describe the hydroxamic acid OH internal rotor potential, leading to predictions for the relative energies in reasonable agreement with experiment. This force field was used to generate a disconnectivity graph for the lowenergy portion of the potential energy landscape of SAHA. This disconnectivity graph contains more than one hundred minima, and maps out the lowest-energy pathways between them, which could then be characterized via DFT calculations.

This

combination of force field and DFT calculations provides insight to the potential energy landscape and how population was funneled into the three observed conformers.

2


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I.

Introduction

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor that binds directly to the catalytic site of the enzyme, thereby blocking substrate access. SAHA is known to inhibit class I and class II HDACs and arrests cell growth of a wide variety of transformed cells.1 SAHA has demonstrated significant anticancer activity in both hematologic and solid tumors.2-3 Receiving approval by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL)4-5 in 2006, SAHA has become the lead compound in a promising new class of anticancer drugs.

Figure 1. Chemical structure of suberoylanilide hydroxamic acid (SAHA).

One of the most striking features of the structure of SAHA is its linear juxtaposition of non-polar aromatic, polar amide, non-polar alkyl chain, and polar hydroxamic acid groups.

The two polar groups contain hydrogen bond donor and acceptor groups,

enabling hydrogen bonds to be formed, either internal to the molecule or with its surroundings, involving both the amide group “head” and the chelating hydroxamic acid group “tail”.

The hexyl chain that links them gives great flexibility to the 3



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molecule in interacting with its surroundings.

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For instance, the crystal structure of

SAHA itself involves an array of “linear” SAHA molecules in which the six-carbon chain is extended in an all-trans structure that enables H-bonds “head-to-tail” and “tailto-head” H-bonds between SAHA molecules in adjacent layers.6 More importantly, in the crystal structure of the complex of SAHA with histone deacetylase like protein (HDLP) complex,7 SAHA also adopts an extended conformation, but this time with the aromatic “head” sitting at the entrance to a long, cylindrical HDLP pocket.

The 6-carbon alkyl chain extends down the length of the

pocket, where its hydroxamic acid “tail” can engage in bidentate chelation to a Zn+2 cation located at the bottom of the polar HDLP pocket. From a fundamental viewpoint, what is not yet established is the inherent conformational preferences of the SAHA molecule in its isolated form. addresses that need.

This work

In the gas phase, the many intermolecular interactions with the

binding pocket or other SAHA molecules are removed, leaving only the molecule’s intramolecular interactions to dictate its inherent conformational preferences.8-10 With one hydrogen bonding group adjacent to ring and the other at the end of its long, flexible C6 hydrocarbon tail, SAHA is able to form “head-to-tail” and “tail-to-head” hydrogen bonds involving the several donor and acceptor sites in the amide and hydroxamic acid groups (Figure 1). potential

presence

With a flexible alkyl chain connecting them, one anticipates the of

several

competing

“head-to-tail”

and

“tail-to-head”

conformational isomers, with isomerization occurring on a potential energy landscape that is prototypical in form. 4


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One might anticipate that “head-to-tail” and/or “tail-to-head” cyclic structures will be low in energy due to the hydrogen bond(s) so formed.

As Figure 1 shows, two

NH···O=C H-bonds are possible that constitute 11-membered rings (denoted as ‘C11’). The OH group offers another H-bond donor site, to which it could bind to the head amide group either alone, or in concert with the hydroxamic acid carbonyl group. These unique bonding arrangements assume that the hexyl chain is able to loop back on itself with minimal conformational strain.

However, in pure alkanes, the extended

‘all-trans’ structure is most stable for alkyl chains up to 18 in length, with each gauche defect destabilizing the structure by about 2 kJ/mol.11 Thus, formation of H-bonded cycles in SAHA of necessity occurs with some conformational strain in the alkyl chain. Furthermore, the sheer number and increased floppiness of extended conformations argues for their dominance at higher temperatures on entropic grounds.

Thus, it is

fascinating to explore the inherent conformational preferences of SAHA in the gas phase. In this paper the conformation-specific infrared (IR) and ultraviolet (UV) spectra of the isolated SAHA molecule are presented, carried out under expansion-cooled conditions in the gas phase. Transitions due to three different conformers of SAHA are observed. Assignments are made for these conformers based on several pieces of spectroscopic data. The conformational specific infrared spectra in hydride and amide I/II regions serve as diagnostics for structural determination of individual conformers. The single-conformation ultraviolet spectra also shed light on the conformations present, due to large variations in the S0-S1 origin transition frequencies. The structures, 5



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relative energies, and harmonic vibrational frequencies for many low lying conformational minima of SAHA have been calculated using electronic structure methods, providing several points of comparison between theory and experiment. Finally, as a means of assessing the over-all form of the potential energy landscape for the molecule, we modify a common molecular mechanics force field to more accurately account for the hydroxamic acid OH internal rotation, and use it to calculate a disconnectivity graph for SAHA.

This pictorial summary of the potential energy

landscape provides a useful means of understanding the observed and ‘missing’ conformations, aided by predictions of the isomerization pathways between them. II.

Methods

A. Experimental methods The experimental methods used in the present study have been described in detail elsewhere.12 SAHA was purchased from Cayman Chemical at 98% purity, and used without further purification.

In the present case, laser desorption was used to vaporize

the sample. The powder sample was rubbed into the surface of a graphite rod to attain a smooth, visually uniform top surface layer. The graphite rod was placed directly underneath the nozzle orifice via a load-lock assembly. A linear actuator (NSC 200, Newport) was applied to move the steel rod linearly to ensure exposure of new sample to the desorption laser. A Nd:YAG laser (Continuum Minilite II) operating at 20 Hz (5mJ/pulse, 2mm beam diameter) was used for desorption and was aligned through a window above the pulsed valve directly onto the graphite rod. Ultra high purity helium and ultra high purity argon were used as buffer gases (2-3 bar backing pressure) in the 6


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supersonic jet expansion, pulsed at 20 Hz out of a pulsed valve (General, Series 9) with a 500 µm diameter orifice. The expansion was skimmed by passing through a conical skimmer placed ~2 cm downstream to form a molecular beam, which was subsequently photoionized in the ionization region of a time-of-flight (TOF) mass spectrometer. Trace water in the sample or gas handling lines led to formation of the SAHA-water complex. Monitoring the SAHA parent mass channel (m/z 264), one–color resonant twophoton ionization (R2PI) was used to record mass selected UV excitation spectra in the S0-S1 region. The collimated, frequency doubled output of a Nd:YAG (355nm) pumped dye laser was used as the ultraviolet light source. Fluorescein 548 and Rhodamine 6G were used in the dye laser to cover the wavelength range from 275 to 281 nm at pulse energies of 0.1-0.3 mJ/pulse at a 20 Hz repetition rate. Conformation-specific IR spectra were taken using resonant ion dip infrared spectroscopy (RIDIRS) in the hydride stretch region (3200-3500 cm-1) and amide I/II (1450-1850 cm-1) regions. In this double resonance method, the IR beam was generated by a Nd:YAG pumped optical parametric converter (LaserVision) and was introduced into the chamber coaxially and counter propagating the UV laser beam. To record a spectrum, the UV laser was fixed on a transition in the excitation spectrum correlated with a single conformer while the infrared laser was turned through the region of interest. The UV laser was pulsed at twice the frequency of the infrared laser, delayed from the IR by 200 ns. When the IR frequency is resonant with a transition which shares the same ground level as the UV laser, the IR pulse will remove a fraction of the ground state population by absorption. 7



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The difference in ion signals between IR “on” and IR “off” was monitored by scanning the IR laser and using a gated integrator (Stanford Research Systems) in active baseline subtraction mode. To generate IR light in the amide I/II regions, difference frequency mixing of signal and idler beams from the OPO was carried out in a AgGaSe2 crystal. IR laser powers were 3-5 mJ/pulse in the amide NH stretch region, and 0.5-1.0 mJ/pulse in the amide I/II region. All RIDIR spectra were recorded by monitoring the origin transition for each conformer in their UV excitation spectra respectively. In order to record conformation-specific electronic spectra, IR-UV hole burning spectroscopy was employed. The method uses an identical configuration to RIDIR spectroscopy, except the wavelength of the IR hole-burn laser was fixed at a unique ground-state vibrational transition of a particular conformer observed in the RIDIR spectra while the UV probe laser was tuned through the wavelength region of interest. For all IR-UV HB spectra, multiple infrared transitions in the RIDIR spectra were checked to make sure the hole burn band was unique to a particular conformation. B. Computational methods The long chain present in SAHA endows the molecule with a high degree of flexibility, which makes it possible to adopt a large number of stable conformations and increases the complexity of its potential energy surface (PES). Early attempts to use the generalized Amber force field (GAFF) as a screening tool to identify low-energy minima for optimization via density functional theory calculation showed that this force field was inadequate to describe the hydroxamic acid functionality.

This is not

surprising, since the hydroxamic acid moiety is not included among the test set of molecules (e.g., amino acids, proteins) used in creating the force field.13

In particular, 8


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the weak stabilization between the OH and C=O groups, forming a 5-membered Hbonded ring, is not correctly described. Figure 2 represents the CNOH dihedral angle scan for one of the simplest hydroxamic acids, N-hydroxypropanamide, based on the standard GAFF force field (dashed line). The potential energy curve is nearly flat at dihedral angles within ±60° of planar.

As a consequence, the CNOH dihedral angle has no strong preference to

maintain planarity of the OH group (dihedral 0°), and this led to artificially low-energy structures in the force field searches in which the tail OH group points out of the O(C=O)-C-N-O plane to accommodate additional stabilizing interactions for the OH group, which in SAHA includes the phenyl ring π cloud. To correct this deficiency in the force field, we fit the CNOH hindered rotor potential to one obtained from a relaxed dihedral scan of N-hydroxypropanamide carried out at the B3LYP-D3BJ /6-31+G(d) level of theory, as illustrated by the blue line in Figure 2. The modified GAFF force field dihedral angle scan result plotted in Figure 2 as the black line better describes the dihedral angle preference close to zero degrees and generates structures with energies in much closer agreement with both DFT predictions and experiment.

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Figure 2. CNOH dihedral angle scan results for N-Hydroxypropanamide.

Armed with this modified force field, we pursued a different strategy than in previous studies10 for screening structures for DFT structural optimization.

Rather than simply

using the force field to locate minima, the generalized Amber force field (GAFF) was used to generate a disconnectivity graph as a visualization tool to display the energies of local minima on PES and their connectivity through transition states connecting them. In such a disconnectivity graph, the end of each branch identifies the energy of a particular conformational minimum.

The nodal points represent collections of

transition states in the prescribed energy window that connect the minima below them. In this way, one can trivially locate the highest energy barrier along the minimumenergy isomerization pathway between any two minima on the graph. The theoretical method for generating disconnectivity graphs has been described in detail elsewhere.14 In brief, the PES was surveyed using the general AMBER force field (GAFF), with atomic charges obtained from the semiempirical AM1 bond charge correction approach.

Local minima on the PES were located using a basin-hopping 10


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algorithm15 within a canonical Monte Carlo simulation carried out by the GMIN 2.0 program of Wales and co-workers.16 We carried out up to 100 basin-hopping steps until the global minimum was found and the step size was adjusted in each Monte Carlo step for an acceptance ratio of 0.5. For each minimum determined by the basin-hopping algorithm, transition states were located by calculating the Hessian in GAFF and walking uphill in both directions along the smallest eigenvalues using a hybrid BFGS/eigenvector-following transition state search. All stationary points were converged to a root-mean-squared gradient of less than 4X10-6 kJ.mol-1 Ǻ-1.

Then the minima connected to the transition states were

identified using the DNEB/L-BFGS method developed by Wales and coworkers.17-19 Previously unknown minima were added to the growing database of minima, transition states, and pathways, which were then used to generate the disconnectivity graph. Finally, we further systematically expanded the tree by increasing the number of connections per minimum through single-ended transition state searches and the overall connectivity of the disconnectivity graph through parallel double-ended transition state searching.

The disconnectivity graph so generated will be presented and discussed

after the experimental data has been considered. Fifty unique conformational minima with lowest energies in the disconnectivity graph were identified and served as starting geometries for further optimizations using density function theory (DFT) calculations using the Gaussian 0920 suite of programs. These calculations involved tight geometry optimizations followed by harmonic vibrational frequency calculations using B3LYP21 or M05-2X22 hybrid functionals with 11



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the 6-31+G(d) basis set. The dispersion correction from Grimme and co-workers with Becke−Johnson damping (D3BJ)23-24 was added to the B3LYP functional to account for the London dispersion energy not correctly described by standard DFT calculations. The order of the relative energies of the conformers was not changed significantly between the M05-2X and B3LYP-D3BJ calculations.

In this paper we use the

B3LYP-D3BJ calculations to report relative energies and calculated harmonic frequencies, as its predictions were closer to experiment than the M05-2X calculations. The harmonic frequency calculations aided in the assignment of conformational isomers observed in the experiment. These frequencies were scaled by 0.96 for free NH stretch, 0.948 for hydrogen bonded NH and OH stretches and 0.985 for amide I/II frequencies. These scale factors were chosen by scaling the calculated IR frequencies of the assigned structure of SAHA conformer A to the experimental frequencies, where the corresponding patterns provided an unequivocal match between experiment and theory. Finally, vertical excitation energies and excited state geometries were computed with time-dependent density functional theory (TDDFT) with the same basis sets as mentioned above. C. Nomenclature As discussed above, since the amide and hydroxamide groups of SAHA are separated by a C6 alkyl chain, several different intramolecular H-bonding arrangements are predicted to be possible in the gas phase by the theoretical calculations. Consequently, the structures are grouped first into families by H-bonding pattern. The NH and C=O 12


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groups adjacent to the phenyl ring are denoted as the head NH and head C=O, while the corresponding groups in the hydroxamic acid are denoted as ‘tail’ groups. The size of a hydrogen bonded ring formed by the NH or OH with the C=O groups is denoted as Cn,25-26 where n stands for the number of atoms involved in the ring. In 38 out of the 50 structures optimized through DFT calculations, the hydroxamic acid C=O and OH groups are cis to one another, thereby forming a C5 ring. At the same time, in 19 of the calculated structures, the head N-H group forms an additional intramolecular hydrogen bond with the tail C=O, forming a C11 ring. This pattern is described as a Head to Tail pattern (labeled as H-T), which are the preferred arrangement in most of the low energy conformers. Since the amide groups at the head and tail are in reverse order to one another, C11 rings can also be formed through intramolecular hydrogen bonds between tail NH and head C=O groups. In total, 9 of the calculated conformers adopt this Tail to Head pattern (labeled as T-H). In 7 calculated structures, the tail NH, instead of adopting a T-H hydrogen bond, points instead to the π cloud of the phenyl ring, forming a weak NH-π intramolecular hydrogen bond (labeled as NH-π ). 3 structures are predicted to form a head NH to tail OH intramolecular hydrogen bond (Labeled as HTOH). For the remaining 12 structures, the C5 rings at the tail are broken and the tail OH group points to the phenyl ring, thereby forming an OH-π intramolecular hydrogen bonds.

Notably, these structures still retain the C11 H-T H-bond as well, and are

thereby labeled as H-T/OH-π.

For all 50 of the calculated structures, the amide group

near the phenyl ring is in a trans-amide arrangement. The possibility for cis-amide arrangement is also explored and will be discussed in detail in Section IV.C. 13



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It is clear from the above description that several conformers of each H-bonding arrangement (H-T or T-H) can be formed, which therefore must differ in the configuration of the alkyl chain that links them.

We therefore designate the full

conformational structure by denoting the alkyl chain dihedral angles along the hydrocarbon chain, numbering the C-atoms C1 to C6 respectively from head to tail. Including the C=O carbons, there are five dihedral angles along the alkyl chain, which are designated by α for C(=O/Head)-C1-C2-C3, β for C1-C2-C3-C4, γ for C2-C3-C4-C5, δ for C3-C4-C5-C6 and ε for C4-C5-C6-C(=O/Tail). As is standard, we will use gauche+ (g+), gauche- (g-), anti (a) and eclipsed (e) to describe dihedral angles around +60°, 60°, ±180° and ±120°, respectively. Thus, the complete name of a structure would contain both H-bonding pattern and C6 chain orientation, for example, T-H (g+,g+,a,g+,g+). III.

Results and analysis

A.

R2PI and IR-UV holeburning spectra

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Figure 3. R2PI (top trace) and IR-UV HB spectra (lower traces) for SAHA. Asterisks in the R2PI spectrum are tentatively ascribed to hot bands of conformer A. Since the electronic chromophore of SAHA is closely related to that in trans-formaniliide (tFA) and trans-acetanilide (tAA), their electronic origins are shown in the figure for reference.29

The top trace of Figure 3 presents the one-color R2PI spectrum of SAHA in the S1 ← S0 origin region, covering from 35590 to 36350 cm-1. Two distinct groups of transitions are present in the spectrum, with a 450 cm-1 gap between them. The intensities of the transitions in the higher wavenumber region are almost three times stronger than those in the lower wavenumber region. Using infrared transitions determined from RIDIR spectroscopy, a series of IR-UV hole burning spectra corresponding to each individual conformation are presented below the R2PI scan in Figure 3. The R2PI spectrum divides into transitions due to four unique structures (AD). The S0-S1 origin transition of conformer A is at 36095 cm-1 (A) and has short FrankCondon progressions involving vibrations of frequency 23 cm-1 and 67 cm-1 built off of it.

The IR-UV hole-burning spectrum of A (recorded with the IR at 3258 cm-1)

accounts for all the major transitions in the blue part of the R2PI spectrum, except the small transitions marked by asterisks, which are tentatively ascribed to hot bands based on changes in their intensity with desorption conditions.

The background present in

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the R2PI spectrum but missing in the hole-burning spectrum, is likely due to higherfrequency vibronic activity arising from B-D. The corresponding S0-S1 origins for B-D are shifted more than 400 cm-1 to the red of A, appearing in close proximity of one another at 35648 cm-1(B), 35675 cm-1(C) and 35654 cm-1 (D), respectively. These spectra also exhibit some Frank-Condon activity in low frequency vibrational modes, as summarized in Table 1. Based on the fact that the S0-S1 origins of B-D differ from one another by no more than 27 cm-1, and the associated hole-burning spectra display similar low frequency vibronic structures, it is clear that the environments for the aromatic ring are similar in all three. This suggests that B-D are all of one structural type while A is of a different type. The frequency positions of the S0-S1 origins for A-D already provide some clue to the structures involved.

The electronic origins of B-D are well red-shifted from that

of trans-formanilide (tFA, 36004 cm-1) and trans-acetanilide (tAA, 35902 cm-1),27 while that for conformer A is to the blue, as shown in Figure 3. Previous studies28 have identified H2O complexes with tFA in which H2O acts either as H-bond donor to the C=O site or as H-bond acceptor from the NH.

Their electronic origins are shifted to

the blue in the former case (36,114 cm-1) and to the red in the latter (35,783 cm-1). Based on these simple arguments, we anticipate that conformer A will be a T-H structure, while conformers B and C are both H-T.

We will see shortly based on their

infrared spectra that this is indeed the case. It is worth noting that, in both tFA and tAA, previous studies have also observed a minor cis-amide conformer (~6% of trans).29 The UV spectrum of the cis-amide 16


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conformer of formanilide has its S1←S0 origin band ~1000 cm-1 red shifted from that of the trans isomer.27 The corresponding transition in acetanilide has not been detected to date.

In the SAHA spectrum, there are several small peaks in the R2PI spectrum in

Figure 3 around 36000 cm-1 (marked by asterisks) that do not burn out with conformer A.

Their weak intensity prevented recording RIDIR spectra of these bands; however,

the balance of evidence points to other these transitions as hot bands of A or water complexes rather than other conformers of SAHA monomer.

Scans taken over the

35590 – 36350 cm-1 region revealed no further transitions not accounted for by A-D. As a result,

experimental evidence points towards all observed conformers arising

from the trans-amide structure of SAHA, the only isomeric form observed in solution or in the crystal.6

We will return to this point in the discussion.

Finally, in the

supplementary material, evidence is presented that structure D is in fact due to a SAHAH2O complex, since the IR spectrum contains two more hydride stretch transitions. The ion signal appears in the monomer mass channel through its efficient fragmentation following photoionization. Our main focus in this paper is on the SAHA monomer, with the SAHA-H2O complex discussed further only in supplementary material. B.

RIDIR spectra of conformers A-C

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Figure 4. RIDIR spectra for SAHA conformer A,B and C in the (a) hydride stretch and (b) amide I/II regions. Calculated IR spectra at the DFT B3LYP D3BJ/6-31+G(d) level of theory are shown below as stick diagrams in black. These frequencies were scaled by 0.96 for free NH stretch, 0.948 for hydrogen bonded NH and OH stretches and 0.985 for amide I/II frequencies. Asterisks indicate infrared transitions used to record IR-UV holeburn scans.

Figure 4 presents a series of RIDIR spectra recorded in the hydride stretch (Figure 4a) and amide I/II regions (Figure 4b) for SAHA conformers A, B, and C. The S1←S0 origin transitions of the three conformers were used as monitor transitions for the RIDIR spectra, resulting in the single-conformer IR spectra shown. The asterisks in the RIDIR spectra denote the transitions used to record the IR-UV HB spectra in Figure 3. The B3LYP-D3BJ /6-31+G(d) predictions for the best-fit vibrational frequencies and peak intensities of the assigned structures are shown as stick diagrams immediately below the experimental RIDIR spectra in Figure 4.

Comparison between observed

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and calculated vibrational frequencies in the hydride stretch and mid-IR are given in Table 1. Since the SAHA molecule possesses one OH and two NH groups, three hydride stretch fundamentals are anticipated in the RIDIR spectrum of each conformer.

In all

three conformers, there is a single free NH stretch fundamental in the 3450-3500 cm-1 region.

While conformers B and C are at similar wavenumber positions (3497 cm-1),

that for conformer A is at 3457 cm-1, some 40 cm-1 lower.

Thus, as with the UV

spectra, conformer A seems to be of one structural type, different from those of B and C.

Based on calculations of the conformations of SAHA, including extended

conformers where both amide and hydroxamic acid NH groups are free, it is clear that the frequency of the free NH stretch fundamentals of head and tail NH groups are indeed different, with the free amide NH about 40 cm-1 higher in frequency than the free hydroxamic acid tail.

This provides a second piece of evidence that conformer A

is a T-H structure, while B and C are both from the H-T family. The RIDIR spectrum of conformer A also contains two hydride stretch fundamentals shifted to much lower frequency (3250-3310 cm-1), which also show an increased intensity and broadening, all of which are signatures of the formation of H-bonds.30-31 Similar intense, broadened, low-frequency transitions are also present in conformers B and C, indicating that all three conformers possess significant intramolecular H-bonds. However, when compared with conformer A, all hydride stretches of B and C are shifted to higher frequencies, consistent with weaker H-bonds in B and C than in A. For conformer B, scans that extend down to 3200 cm-1 revealed no additional transitions, 19



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Page 20 of 47

suggesting that the broadened band at 3351 cm-1 may arise from an overlap of two Hbonded hydride stretches32 that are more closely spaced than in conformer C. In the amide I (1650-1750 cm-1) region, two transitions are resolved for all conformers, corresponding to the two C=O stretches in the molecule.

The 1450-1600

cm-1 region is somewhat more complicated, containing transitions due to the NH bends of amide and hydroxamic acid groups, and several weak benzene CH bend fundamentals.

Again, the patterns for conformer B and C resemble each other while

the pattern for conformer A is different. The sharp transitions in all three spectra around 1620 cm-1 are due to aromatic C=C stretching modes.

Figure 5. Calculated optimized structures assigned for SAHA conformers A to C and structure D, assigned to the SAHA-H2O complex, at the DFT B3LYP-D3BJ/6-31+G(d) level of theory.

The zero-point corrected relative energies are included. 20


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Comparison of the experimental spectra with the scaled harmonic frequencies and IR intensities of low-energy conformers of SAHA (stick diagrams in Figure 4) leads to the structural assignments for conformers A-C presented in Figure 5. The match between experiment and calculation, relative to alternatives (see Figure S4 and Table S1), is sufficiently good to make these assignments secure.

As anticipated,

conformers adopting both H-T and T-H patterns are observed in the gas-phase. Table 1 compares the observed and calculated vibrational frequencies for conformers A-C in the hydride stretch and mid-IR regions.

Low-frequency vibrations

that appear in the R2PI spectrum are also compared with the assigned structures, under the assumption that electronic excitation will change these vibrational frequencies only modestly from their ground state values.

This latter comparison adds confirming

evidence to the assignments, but couldn’t be considered diagnostic on its own.

Table 1. Summary of calculated (Calc) and observed (Obs) vibrational frequencies and assignments of SAHA monomer and the SAHA-H2O complex, calculated at the DFT B3LYP-D3BJ/6-31+G(d) level of theory. Torsional modes (cm-1)a

Hydride stretches (cm-1)b

ν2

ν1

ν tail NH

ν OH

ν head NH

Molecule

Obs

Calc

Obs

Calc

Obs

Calc

Obs

Calc

Obs

Calc

SAHA A

23

23

67

66

3256

3259

3306

3310

3457

3459

SAHA B

36

31

64

70

3497

3492

3351

3342

3351

3335

SAHA C

33

34

3497

3490

3373

3348

3390

3352

21



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a

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Unscaled harmonic frequencies and experimentally observed spacings of Franck-

Condon activity from the IR-UV holeburning scans. b

Hydride stretch fundamentals are scaled by 0.96 for free NH stretches, 0.948 for

hydrogen bonded NH and OH stretches, respectively.

See text for further discussion.

Conformer A is assigned to a T-H structure (Figure 5A) labelled as T-H (g+,g+,a,g+,g+).

This structure is also the calculated global minimum at the B3LYP-

D3BJ/6-31+G(d) level of theory, consistent with the large intensity of its transitions in the UV spectrum (Figure 3).

The free head NH stretch of A appears at 3457 cm-1,

similar to the NH stretch fundamentals of trans-formanilide (3463 cm-1) and transacetanilide (3472 cm-1).33 The slight shift to lower wavenumber is potentially due to anti-cooperativity8 in which the head NH bond is weakened by formation of an intramolecular H-bond to its amide C=O group, which acts as an acceptor in the T-H intramolecular H-bond, forming an 11-membered H-bonded ring (labeled ‘C11’, Figure 5).

The tail-to-head intramolecular H-bond is short (1.90 Å, Figure 5A),

producing a tail NH stretch fundamental at 3256 cm-1. The tail OH group of conformer A is cis to the C=O group, forming a C5 intramolecular H-bond with the C=O oxygen, which places the OH stretch fundamental at 3306 cm-1. In the amide I region, the head C=O is acceptor for the C11 T-H hydrogen bond. As a result, its C=O stretch fundamental is shifted down in frequency to 1697 cm-1 when compared to that of trans-fromanilide (1742 cm-1) and trans-acetanilide (1728 cm-1). The tail C=O of the hydroxamic acid group appears at 1681 cm-1 due to the unique chemical environment of this group and the C5 ring it forms with the tail OH.

The 22


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amide II band for the head NH group is at 1540 cm-1, slightly higher in frequency than in trans-formanilde (1529 cm-1) and trans-acetanilide (1528 cm-1), reflecting the same indirect effect of the strong H-bond to its C=O group. For the tail NH, the predictions of the calculations are that its NH bend fundamental should be shifted to even higher frequency (~1570 cm-1). However, this transition is predicted to have very weak intensity and wasn’t observed experimentally. The structure assigned to conformer B (Figure 5B) is calculated to be 5.1 kJ/mol higher in energy than conformer A at the B3LYP-D3BJ/6-31+G(d) level of theory. Notably, this structure is determined to be the lowest-energy member of the H-T family. Conformer C, with its many spectral similarities to B, is assigned to a conformer in the same family, shown in Figure 5C, with an energy only 1.2 kJ/mol higher than B.

The

difference between these structures lies largely in the folding of their C6 alkyl chains, as reflected in their dihedral angles, with B labelled as H-T (g+,g+,e,a,g-) and C labelled as H-T (a,g-,g-,a,g-). In general, conformer B adopts a tighter loop for the C6 hydrocarbon chain while a somewhat more extended chain is favored by conformer C. This leads to a slightly larger distance between the head NH and tail C=O groups in C than B, and a different approach angle for the NH…O=C H-bond. As anticipated, for both conformer B and conformer C, the free tail NH stretch fundamentals appear at around 3497 cm-1, about 40 cm-1 up from the position of the free head NH stretch observed in conformer A.

The tail OH retains the same structural

preference in forming a weak C5 H-bond with the tail C=O. However, in this H-T family, the tail C=O also acts as an acceptor to the donor head NH group in the H-T H23



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Page 24 of 47

bond, forming a bifurcated double ring structure with both C5 and C11 H-bonds sharing the same tail C=O group. As a result, both H-bonded tail OH and head NH stretches are shifted up in frequency relative to those in A, revealing another anti-cooperative effect, in that one H-bond to the same acceptor group weakens the other. For conformer B, as the calculated stick diagram suggests, those two transitions are overlapped with each other and thus forms a broadened peak at 3351 cm-1. According to the calculation, the C5 tail OH is much weaker than the H-bonded head NH stretch. In conformer C, calculations predict that the relative wavenumber positions of the H-bonded NH and OH stretch are reversed, appearing at 3390 cm-1 and 3373 cm-1 respectively. In the amide I region of both B and C, the tail C=O stretch is shifted to lower wavenumber relative to A, reflecting its character as a double H-bond acceptor. The head C=O stretch, which is now free, shifts up to around 1724 cm-1, similar to the value in trans-acetanilide (1728 cm-1).

As expected, the head NH bending

fundamentals are also shifted slightly up in frequency to 1560 cm-1 for both conformers in the amide II region as a result of engaging in a H-T intramolecular H-bond. In arriving at the assignments, a large number of alternative low energy structures and structures in other conformational families were compared with experiment, including those engaged in NH-π, H-TOH and H-T/OH-π H-bonded architectures (Figure S4). However, they were either completely inconsistent with experimental patterns, or were significantly poorer matches. In addition, most of the structures belonging to different families suffer from significantly higher energies than the assigned structures, consistent with their absence in the expansion. 24


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IV.

Discussion

A. Inherent conformational preferences of SAHA monomer A primary motivation for the present study was to understand the inherent conformational preferences of SAHA monomer in the gas phase, where environmental effects are removed. Whether in crystalline form or in its binding to the HDAC enzyme pocket, the alkyl chain is extended in order to facilitate interactions with other SAHA molecules or the binding pocket.

Yet, in the absence of these environmental effects,

the C6 alkyl chain, which prefers an extended conformation, is countered by the stabilizing effect of intramolecular H-bond(s) between the molecule’s “head” amide group and “tail” hydroxamic acid. Our study seeks to determine how many and which conformers are present in the gas phase.

Using laser desorption to bring SAHA into the gas phase, and cooling the

molecules in a supersonic expansion, we have recorded single-conformation UV and IR spectra that led to the identification and assignment of three conformers of SAHA monomer, shown in Figure 5.

All three conformers are tightly-folded structures that

contain intramolecular H-bonds between “head” amide and “tail” hydroxamic acid groups. 1

The unique wavenumber positions of the free NH groups of head (3457 cm-

) and tail (3497 cm-1) provide characteristic spectroscopic signatures of H-T and T-H

structures.

In all three conformers, the hydroxamic acid OH group is cis to the C=O

group, forming a 5-membered H-bonded ring (C5) that is opposite to its orientation when chelating the Zn+2 cation in the HDAC binding pocket, which uses the oxygen lone pairs of the C=O and OH groups.7 25



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Page 26 of 47

In conformer A, the global minimum, the hydroxamic acid tail engages as H-bond donor via its NH group to the C=O group of the amide head, forming an elevenmembered H-bonded ring (C11) in a T-H structure.

This H-bond is strong, with a H-

bond length of 1.90 Å, leading to a broad and intense NH stretch fundamental at 3256 cm-1.

In conformers B and C, the direction of the H-bond is reversed, with the

molecule’s head NH acting as H-bond donor to the tail C=O group.

These T-H

hydrogen bonds also form C11 rings due to the reversal in order of the NH and C=O groups in the amide and hydroxamic acid moieties of SAHA.

Conformers B and C

have R2PI transitions about one-third the size of those of A, consistent with their calculated relative energies about 5 kJ/mol higher than A. They also have somewhat weaker head-to-tail H-bonds in the 3350-3400 cm-1 region, with calculated hydrogen bond lengths of 2.11 and 2.14 Å, respectively. Each of these structures incorporates a turn in the alkyl chain.

In previous studies

from Luttschwager et al.,11 Raman spectra provided spectral evidence that the straightchain n-alkanes prefer an extended structure up to n=17-18, with each gauche defect providing a destabilization of ~2 kJ/mol.

For pure alkyl chains longer than this

threshold length, the alkyl chain folds back on itself using a turn composed of four gauche ‘defects’, configured as (gm-2, gm-1, a, gm+1, gm+2).

By positioning this turn mid-

way through the alkyl chain (m=n/2), the two all-trans segments on either side are antiparallel to one another, where dispersive attractions can act in concert along these segments to stabilize the folded structure.

26


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In SAHA, the 6-carbon alkyl chain would by itself energetically prefer an extended structure.

However, as the labeling scheme in Figure 5 indicates, the presence of the

amide and hydroxamic acid groups at either end of the alkyl chain cause it to fold into a 4-gauche (conformer A), 3-gauche (conformer C), or 3-gauche, 1-ecclipsed (conformer B) turn that positions the tail and head groups where they can engage in a H-bond that stabilizes the fold.

The turn in A is just as in the pure alkyl chains, in this

case (gα, gβ, a, gδ, gε), and this ideal turn does indeed position the head and tail for a strong, near-linear T-H hydrogen bond.

In Table 2, we have listed not only the

dihedral angles along the alkyl chain (α−ε) but those on either side (NC12 and 56CN), which denote the orientation of the first and last C-C bond relative to the amide or hydroxamic acid planes.

It is noteworthy that the alkyl chain prefers an out-of-plane

orientation for these two ancillary dihedral angles.

The end result is that the amide

and hydroxamic acid planes are nominally perpendicular to one another in all three conformers (Figure 5), with different approach angles for the H-bond so formed.

Table 2.

Dihedral angles (degrees) along the C6 alkyl chain of the three observed

conformers of SAHA monomer from head-to-tail.

Conformer A B C cis-1 cis-2

NC12 +86 -130 -95 +130 +144

Dihedral Angle (degrees) α β γ δ C123 1234 2345 3456 +65 +75 -164 +72 +62 +56 -111 +164 +165 -58 -58 +164 -79 +62 +64 -179 -44 -46 +171 -63

ε 456C +61 -65 -67 +59 -64

56CN -118 +131 +133 +63 +126

27



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B.

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Disconnectivity graphs, isomerization pathways, and observed conformers Single-conformation IR and UV spectroscopy provides a powerful tool for dissecting

a complicated spectrum into its constituent components due to individual conformational isomers.

Assignments are made by comparing the observed single-

conformer spectra with ab initio or DFT calculations.

In order to make these

assignments, however, classical force field searches are typically used as a screening tool to locate conformers, and to order them by force field energy as a means of prioritizing the quantum chemical calculations, which are much more computationally intensive. The field of single-conformation spectroscopy is now at a point where it is capable of serving as the basis for refining these force fields, especially in their applications to isolated, gas phase molecules, using the assigned structures as benchmarks for doing so.

Accurate force fields would then open up whole new classes of problems for

exploration, both in the size of the molecules which could be explored, and in going beyond spectroscopy to understand the dynamics of conformational isomerization.34 In this latter context, the disconnectivity graph serves as a powerful tool for summarizing the entire potential energy landscape for the molecule, with all its conformational minima, transition states, and linked pathways between individual minima. In the present work on SAHA, we have taken steps in this direction, first in modifying the hydroxamic acid CNOH dihedral potential within GAFF based on B3LYP-D3BJ/631+G(d) calculations, as shown in Figure 2.

Then, armed with these parameters, we

used the modified version of GAFF to create a disconnectivity graph for gas-phase

28


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SAHA monomer, shown in Figure 6.

We were motivated to construct the

disconnectivity graph for SAHA because of the prototypical nature of the conformational landscape, with H-T, T-H, and extended conformers all possible, with their interconversion pathways and their energetics difficult to intuit.

Figure 6. (a) Disconnectivity graph for SAHA using the modified general Amber force field (GAFF). Red asterisks indicate the locations of SAHA A and SAHA B. (b) Closeup view of the dashed rectangle region of the SAHA disconnectivity graph where the assigned structures for SAHA A and SAHA B are located.

The zero-point energy

corrected relative energies calculated at the DFT B3LYP-D3BJ level of theory are also indicated (X), taken from Table 3.

29



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Page 30 of 47

Figure 6(a) shows an overview of the GAFF disconnectivity graph for SAHA, while Figure 6(b) focuses in on the section enclosed by a dotted red line, where conformers A and B reside. The disconnectivity graph places stable conformational minima at the end of vertical branches that denote their relative energies.

These minima are

connected by branches that group together transition states of the same energy, grouped into energy bins that are user-determined.

In the present case, transition states are

grouped into bins separated by 1.00 kcal/mol (4.18 kJ/mol).

The numbering of the

conformational minima denotes the order in which they were found in the search process. Note, first, that conformers A, B, and C are all predicted by the modified GAFF force field to be among the low-energy conformers.

This gives confidence that the over-all

structure of the graph is meaningful, and that it can aid a deeper understanding of the conformational landscape of SAHA.

At the same time, the relative energies

calculated by the force field are not in perfect agreement with DFT B3LYP-D3BJ calculations, as can be seen from the positions of the symbols (X) and Table 3. Therefore, in the discussion that follows, we will use the disconnectivity graph to provide an overview of the potential energy surface and isomerization pathways, but use the DFT B3LYP-D3BJ/6-31+G(d) calculations to refine our arguments.

Table 3

contains relative potential energies, both with and without zero-point correction, and relative free energies calculated at 300 K. In order to focus our analysis on the lowest-energy conformations and pathways, the disconnectivity graph in Figure 6(b) is restricted to minima connected to transition 30


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states within 33 kJ/mol of the global minimum, which captures the pathways connecting A, B, and C.

In total 159 minima were identified for SAHA, connected by

207 transition states. One of the insights to be gained from the disconnectivity graph is the way in which it divides the potential energy surface into basins containing sets of low-lying minima connected by transition states that are also comparatively low in energy.

All the low-

lying minima on the potential energy surface have an intramolecular H-bond, whether NH…O=C H-T or T-H H-bonds, and/or those involving a π H-bond (e.g., OH…π). The fully-extended structure in which the alkyl chain is all-trans is calculated to be 44 kJ/mol higher than the global minimum in the GAFF force field, 27 kJ/mol from DFT B3LYP-D3BJ/6-31+G(d).

In this sense the conformational minima that have no

interactions between SAHA’s head and tail are just off the top of the disconnectivity graph in Figure 6. In SAHA, the low-energy isomerization pathways involve breaking/re-forming Hbonds, and reconfiguring the alkyl chain to bring the head and tail groups into contact with one another. Our initial expectation was that the disconnectivity graph would have two major basins involving H-T and T-H minima separated by a comparatively large barrier.

However, this is not the case.

In actuality, the energetics of breaking H-

bonds and reconfiguring the alkyl chains are similar in size, leading to a disconnectivity graph with appearance more like a banyan tree,35 with many low-lying minima separated by barriers of approximately the same height. We use the disconnectivity

31



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Page 32 of 47

graph to understand why conformers A, B, and C appear in the expansion-cooled gas phase sample, and why other low-lying minima are absent. According to the GAFF force field, the SAHA minima are grouped more by having common or similar dihedral angle patterns along the C6 hydrocarbon chain rather than via their intramolecular H-bonding patterns.

This is immediately evident from Figure

6(b), where conformers A and B, which are T-H and H-T structures, respectively, are in the same branch inside the red-dashed rectangle.

This branch also contains the

GAFF global minimum (Min52), while SAHA C, also a H-T structure like B, is in a separate branch with a different dihedral angle pattern. According to GAFF, the global minimum for SAHA monomer is Min52, a conformation that adopts the same dihedral angle pattern (g+,g+,a,g+,g+) as SAHA A, but with a H-T rather than T-H H-bond. isomerization between A and Min52 occurs.

An obvious question, then, is how the Notably, after DFT re-optimization at

B3LYP-D3BJ/6-31+G(d) level of theory, SAHA A becomes the global minimum on the disconnectivity graph, with Min52 calculated to be 2.1 kJ/mol higher in energy. The lowest-energy pathway between Min52 and SAHA A identified by the GAFF disconnectivity graph was recomputed by DFT methods, and is shown in Figure 7. The transition states between individual minima were confirmed by intrinsic reaction coordinate (IRC)36 calculations. Note that the highest energy barrier between Min52 and A is 13.1 kJ/mol, a barrier small enough that population can be funneled from Min52 to A during the collisional cooling process in the early portions of the expansion. Note that the isomerization from Min52 to Min56 involves breaking the H-T hydrogen 32


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bond by reorienting the tail hydroxamic acid group, with the C5-C6-C(=O/Tail)-N(H/Tail) dihedral angle changed in so doing from +103° to -122° in the intermediate state (Min56).

In the second step, the entire formanilide ‘head’ rotates about its N(-

H/Head)-C(=O/Head)- C1-C2 dihedral angle from -141° to 85°, forming a short (1.83 Å) tail-to-head intramolecular H-bond in SAHA A. The second barrier (TS297) is located only 6 kJ/mol above the intermediate state. Thus, the pathway from Min52 to A involves breaking the H-T and forming the T-H H-bond without changing the alkyl chain configuration, and does so over a surprisingly small barrier.

In fact, Rice-

Ramsperger-Kassel-Marcus (RRKM) rate constants for both steps are calculated to be around 1010-1011 s-1 at the average internal energy of SAHA monomer at 300 K ( = 46 kJ/mol), much faster than the cooling rate. So it is anticipated that the population of Min52 originally present in the laser-desorbed monomer is largely interconverted to SAHA A in the supersonic jet expansion and is thus absent in the R2PI spectrum. Similar arguments can be used to rationalize the absence of conformers Min56/139 and Min42/107, which are in the same sub-branches as SAHA A and SAHA B, respectively (Figure 6b). As Table 3 shows, these four minima have very low isomerization barriers (4-5 kJ/mol) to the lowest energy minima in their sub-basin (A or B). Thus, like Min52, structural relaxation into A or B through interconversion over low energy barriers is postulated to account for their not being observed experimentally.

33



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Figure 7.

Page 34 of 47

Stationary points along the lowest-energy isomerization pathway predicted

by GAFF between SAHA A, B, and C, calculated at the DFT B3LYP-D3BJ/6-31+G(d) level of theory. Molecular geometries and optimized parameters for stationary points are included in the supplemental material.

For SAHA B, although it resides in the same general branch of the disconnectivity graph as SAHA A, with the same dihedral angle pattern at the beginning of the C6 hydrocarbon chain, the differences in the last three dihedrals places the two conformers in different sub-branches with a larger isomerization barrier that likely hinders their interconversion.

The barrier from B to A is predicted to be 26 kJ/mol by DFT (Figure

7), slightly higher (~3 kJ/mol) than the barrier between SAHA B and SAHA C molecules. Both the A→B and B→C isomerization pathways are included in Figure 7, and were verified by intrinsic reaction coordinate (IRC) calculations.

34


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The other way in which the populations of the conformers could be modified is if their free energy corrections are quite different from one another. Table 3 also includes the relative free energies of the 10 lowest energy conformers of SAHA at the B3LYPD3BJ/6-31+G(d) level of theory.

Free energy corrections were made at 300 K, not

knowing the internal energy of the laser desorbed molecules prior to cooling in the expansion. Note first that the relative free energies for A, B, and C are three of the five lowest, consistent with their large populations in the expansion.

In free energy, they

are matched only by Min52 and Min139, which have already been argued to lose their population during the collisional cooling process due to small barriers to A.

Indeed,

the negative free energy correction for conformer C is consistent with its presence among those observed. Only two of the seven non-observed minima in Table 3 lack a low-energy cooling pathway to A-C.

Of these Min373 incorporates a H-T intra-molecular H-bond like

that in B and C; however, the tail OH, instead of engaging in a C5 ring with the tail C=O group, points to the phenyl ring and forms an additional OH…π intramolecular Hbond.

This conformer has an energy only 4 kJ/mol above the global minimum and a

free energy close to SAHA B.

With a high isomerization barrier to assigned

conformers (>20 kJ/mol), it is less likely that population initially in this structure would be lost by collisional energy transfer.

Min42 is somewhat higher in both ∆E (8.6

kJ/mol) and ∆G (5.0 kJ/mol), and thus less of a concern.

The present data cannot

determine whether inaccuracies in the calculated relative energies or barrier heights, or some anomaly of the laser desorption process led to their not being detected. 35



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Page 36 of 47

Table 3. Calculated relative energies and free energies of 10 lowest energy conformers of SAHA in the disconnectivity diagram at B3LYP-D3BJ/6-31+G(d) level of theory. Assigned structures are marked in bold. The two lowest energy cis-SAHA structures are added at the end of the table below the dashed line.

ΔE (kJ/mol)

a

B3LYP-D3BJc

ΔG

Free energy correction (kJ/mol) d

(kJ/mol) e

Isomer

GAFF a

B3LYP-D3BJb

SAHA A

7.5

0.0

0.0

0.0

0.0

Min52 (cool to A)

0

2.4

2.1

2.9

5.0

Min373

3.9

3.9

3.6

6.1

9.7

Min56 (cool to A)

9.5

7.6

6.9

3.8

10.7

SAHA B

7.2

7.9

7.3

1.7

9.0

Min139 (cool to A)

5.9

8.7

7.7

0.7

8.4

Min74

6.5

9.0

8.6

4.7

13.3

Min42 (cool to B)

3.8

11.1

8.7

1.5

10.2

Min107 (cool to B)

10.2

9.5

8.8

5.2

14.0

SAHA C

4.1

9.9

8.9

-2.7

6.2

cis-SAHA1

12.5

-8.3

-6.7

12.3

5.6

cis-SAHA2

16.8

-4.6

-2.9

12.7

9.8

General Amber force field 36


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b

Calculated relative energies (kJ/mol) at the B3LYP-D3BJ/6-31+G(d) level of theory,

without harmonic zero-point energy correction. c

Calculated relative energies (kJ/mol) at the B3LYP-D3BJ/6-31+G(d) level of theory,

including harmonic zero-point energy correction. d

Gibbs corrections calculated at pre-expansion sample temperatures (taken as T=300 K)

at the B3LYP-D3BJ/6-31+G(d) level of theory. ΔG=free energy correction+ΔE(B3LYP-D3BJ)

e

The significant free energy corrections of the low-lying conformers listed in Table 3 raises the question of whether these free energy corrections are sufficient to bring the fully-extended conformers into contention with those in Table 3.

To test this

possibility, free energy corrections were calculated for a set of extended-chain conformers. In every case, their internal energies are so high (>25 kJ/mol at B3LYPD3BJ/6-31+G(d) level of theory) that the final ΔG value (>15 kJ/mol) is still much higher than the assigned conformers. As a result, these extended chain conformations of SAHA are not observed in our experiment, as would be obvious from their two free hydride stretch fundamentals. C.

Cis-amide structures and laser desorption

The discussion in Section IV.B neglected one possibility that must still be considered. The head group of SAHA is an alkylated formanilide, Ph-NH-CO-R.

The amide

group’s conjugation with the phenyl ring produces a smaller than typical energy difference between trans-amide and cis-amide isomers, and in formanilide itself, both 37



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cis and trans isomers were observed in the expansion.27

Page 38 of 47

As a result, it was important

also to explore this possibility in SAHA, where the cis-amide structure could potentially engage in hydrogen-bonding arrangements with the hydroxamic acid tail that are not available to trans-SAHA.

However, the standard force field parameters in GAFF

places an artificial barrier at the cis-amide configuration, since in peptides, trans-amide structures are preferred over cis nearly exclusively.

As a result, we explored the cis-

amide conformational space by fixing the amide group dihedral angle in the cis configuration and carrying out a force field search that identified over 400 cis-amide minima for SAHA.

Subsequent optimization of the low-energy structures at the DFT

B3LYP-D3BJ/6-31+G(d) level led to the identification of two conformers shown in Figure 8(a), which are calculated to be lower in potential energy and free energy than any trans-amide SAHA structure so far considered.

All other cis-amide minima are

significantly higher in energy than these two structures.

38


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Figure 8.

(a) Structures, labels, and zero-point energy corrected energies (relative to

SAHA A at the DFT B3LYP-D3BJ/6-31+G(d) level of theory) of the two low-energy conformers of cis-amide SAHA.

(b) Stick diagrams of the calculated frequencies and

IR intensities in the hydride stretch region, using the same scale factors (0.948) for hydrogen bonded NH and OH stretches. The experimental RIDIR spectra in the hydride stretch region are presented in Figure 4(a).

Both these conformers engage in a pair of strong, bridging H-bonds, T-H NH…O=C and H-T NH…OH, with different alkyl chain configurations.

The cis-1 conformer has

shorter H-bonds (1.99 Å H-T, 1.89 T-H) than cis-2 (2.02 Å H-T, 1.95 Å T-H), consistent with its lower energy.

Both these conformers have calculated IR spectra 39



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(Figure 8b) that are completely inconsistent with any of the observed conformers A-C, since no free NH group is present in them, and both NH stretch fundamentals are shifted below 3350 cm-1. We have searched over the 35590 to 36700 cm-1 region in R2PI for additional transitions not yet accounted for.

The TDDFT calculations at B3LYP D3BJ/6-

31+G(d) level of theory predict that cis-1 will have its S1←S0 origin slightly blue (+108 cm-1) of SAHA A, but IR-UV hole-burning proved that all transitions blue of the SAHA A origin are due to A.

Thus, there is no experimental evidence for cis-SAHA

structures in the expansion. Furthermore, the barrier to interconversion from cis to trans is characteristically high (>94 kJ/mol), so that interconversion during collisional cooling is out of the question. With no experimental evidence for cis-SAHA in the expansion, we seek an explanation for their absence.

It seems most likely to us that the laser desorption

process produces only trans-amide structures. As a crystalline solid, SAHA exists exclusively in the trans-amide form. Since laser desorption occurs out of a thin film of the solid directly into the gas phase, and is done under gentle conditions designed not to decompose the sample, we postulate that the initial internal energy of the desorbed SAHA molecules is well below the barrier to trans→cis isomerization.

As a result,

no cis-amide conformers are formed, and full thermal equilibrium of the gas phase molecules does not occur.

As a result, the SAHA monomer is a clear example in

which the observed conformers are affected by the means used to bring them into the gas phase. 40


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V.

Conclusions The present study of the conformational preferences of the gas-phase SAHA

molecule has revealed the presence of three conformers under jet-cooled conditions, all of which are tightly-folded conformers involving tail-to-head or head-to-tail H-bonds. This is in striking contrast to the extended structure SAHA takes up in crystalline form, or when binding to histone deacetylase (HDAC) in its function as anti-cancer drug. The characteristic frequencies of the free NH groups of the amide head and hydroxamic acid tail provide a clear diagnostic of structural type.

The 6-carbon alkyl chain that

serves as chemical linkage is sufficiently long that several structural turns can be formed by it that bring the hydroxamic acid functional group(s) into close spatial proximity with the head amide group. A disconnectivity graph created using GAFF provides insight to the over-all shape of the potential energy landscape for SAHA.

The basins on the potential energy

surface have similar alkyl chain conformations, and interconversion pathways from HT to T-H structures can occur by twisting the head amide and tail hydroxamic moieties successively through hindered rotations that break and then reform new H-bonds between them without reconfiguring the alkyl chain.

The calculated barriers for doing

so are somewhat smaller than the energetic cost for breaking a H-bond in the absence of other compensating attractions.

Alkyl chain reconfiguration is more energetically

costly, preventing isomerization between A, B, and C during the cooling in the expansion.

Calculations predict that two cis-amide structures have potential and free

energies below any of the trans-amide structures, but no experimental evidence exists 41



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Page 42 of 47

for their presence in the expansion. We postulate that the laser desorption of the solid, in which SAHA exists exclusively in a trans-amide configuration, leads only to transamide structures in the gas phase, which cannot interconvert to cis-amide on the timescale of the experiment. While we have not focused attention on it here, we have also observed a single structure for the SAHA-H2O complex (see supplementary material).

This is the first

step in what could be a useful follow-up study to probe the evolution in the conformational preferences of SAHA with increasing number of water molecules in SAHA-(H2O)n clusters.

It could also be useful to search for cis-amide structures when

SAHA is dissolved in non-polar solvents.

Associated Content

Supplementary Information: RIDIR spectra of the SAHA-H2O complex (structure D). Effect of water on the conformational preferences of SAHA. Figure S1 to Figure S4. Table S1. Molecular geometries and optimized parameters for stationary points. This material is available free of charge via the Internet at http://pubs.acs.org

Author Information Corresponding Author *Author to whom correspondence should be addressed:

[email protected]

42


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Acknowledgements. The authors gratefully acknowledge support for this work from the National Science Foundation (NSF CHE-1465028).

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Table of Contents Graphic

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