The document summarizes research on the synthesis of α-fluoromethylhistidine di-hydrochloride (α-FMH), a potent inhibitor of the enzyme histidine decarboxylase (HDC). It describes past methods for synthesizing α-FMH, outlines a novel and efficient synthesis developed by the researchers, and discusses plans to use molecular docking to study the binding of α-FMH to HDC and identify new HDC inhibitors through virtual screening.

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The efficient synthesis of α-fluoromethylhistidine di-hydrochloride, its interaction with HDC,
and the structure-based identification of novel HDC inhibitors
Kelly Considine, Dr. Karl Grozinger, and Dr. Joseph Audie
Sacred Heart University 5151 Park Ave. Fairfield, CT 06825
Abstract
Introduction
Results and Discussion (Continued)
Future Work
References
Methods (Continued)
Acknowledgements
Conclusion
Histidine decarboxylase (HDC) is an enzyme that converts histidine to
histamine. Inhibition of HDC has many medical applications including tumor
suppression, ulcer therapy, circadian rhythm modulation, and allergy
relief. Moreover, HDC inhibitors are needed to study histidine metabolism. α-
fluoromethylhistidine di-hydrochloride (α-FMH) is an extremely potent HDC
inhibitor. Unfortunately, α-FMH is commercially unavailable. Here we report
on the novel, inexpensive, and efficient synthesis of α-FMH, opening the door
to commercially available α-FMH. To study the irreversible binding of α-FMH,
we carried out covalent docking studies on HDC homology models. In an effort
to identify novel inhibitors of HDC, we will perform virtual screening using the
same homology models. To the best of our knowledge, this will be the first
virtual screen applied to HDC. The present study makes an important
contribution to the science of HDC inhibition and suggests new avenues for
studying and therapeutically modulating HDC.
α-fluoromethylhistidine di-hydrochloride (C7H10FN3O2·2HCl ) is one of
the most active histidine decarboxylase inhibitors, but it is not commercially
available (19). Histidine decarboxylase converts histidine to histamine (25).
Histamine: Histamine acts as a neurotransmitter in memory, appetite,
circadian rhythm regulation, intracellular communication, and is a modulator
of cell growth (23). Histamine is involved in allergies, gastric acid secretions,
smooth muscle contraction, vasodilation, inflammatory diseases, bone loss,
tumor progression, learning deficiencies, and epilepsy (17, 18).
Histidine decarboxylase inhibitors: α-FMH has pharmacological actions due to
enzyme inhibition and potential therapeutic uses (19). It can control certain
tumors that have a high histamine level, act as a therapy for ulcers and allergy,
increase slow-wave sleep, decrease motor-excitation, decrease learning and
memory potential, treat inflammatory diseases, neurological and
neuroendocrine diseases, osteoporosis, and neoplasias, and prevent certain
inflammatory responses, peptic ulcers, and schizophrenia (17, 19, 21, 22).
Chemical bonds in α-FMH: Replacement of hydrogen by fluorine should not
have a major consequence on steric hinderance. Fluorine is the most
electronegative element making a polar bond with carbon creating a
hydrophobic molecule that more easily crosses the lipid membranes, in order
to act on the appropriate enzyme.
Ring opening of aziridines: Aziridines have a ring strain of 26.7 kcal/mol which
shifts the reactivity to ring opening, however, when inactivated they are not
highly reactive towards nuclear attack (7). It is predicted that aziridine 1,
scheme 1, will demonstrate ring opening at the least hindered position.
α-fluoromethylhisitidine synthesis: Various patents describe the preparation
of α-FMH, but they are not suitable to scale-up because they are low yielding
and not cost effective. One reagent used to fluorinate amino acids was HF
containing BF3 or SbF5 (12, 13). Other patents describe the fluorination of α-
amino alkanoic esters to yield α-FMH (14, 15). A synthesis was reported using
Seebach’s chemistry of self-regeneration of stereocenters. L-histidine methyl
ester was converted to 1,3-imidazolidinones which was reacted with CH2ClF, a
green house gas (no longer available), to give α-FMH (10).
Molecular docking: There is no definitive active site defined in the literature,
so this will allow us to predict a binding site to use in virtual screening studies.
Kelly Considine acknowledges the financial support from Sacred Heart
University in Fairfield, CT and Boehriniger-Ingelheim Parmaceutical Inc. for the
donation of aziridine (6).
Karl Grozinger acknowledges the valuable support from the Chemical
Development Department at Boehringer-Ingelheim Pharmaceutical Inc. (Victor
Fuchs, Matt Hrapchak, and Jon Lorenz).
Any future research on the synthesis would be to identify a scheme
that allows for aziridine ring opening while preventing dimerization. In
addition, a synthesis that proceeds without racmeization would be ideal.
A goal of the docking study is to put the covalent bond in between α-FMH and
the appropriate serine residue and to further perform monte carlo
minimizations.
After finding the appropriate model with the lowest energy, or possibly
several predicted models, the next step in this project is to use virtual
screening to suggest better small molecule inhibitors for histidine
decarboxylase as potential drug leads. Also, docking of histidine methyl ester,
another known inhibitor, would help to determine the best inhibition.
Furthermore, an enzyme assay would be beneficial in order to
determine the enzymatic inhibition.
The results here indicate that the synthesis produced a moderate yield
of α-FMH at 57%. It was determined that the best yield was achieved when
using 10 equivalents of HF/pyridine to the 1 equivalent of methyl 2-aziridinyl-
3-(N-triphenylmethyl-4-imidazolyl) propionate, but 5 equivalents is sufficient.
This was a novel synthesis that was cost effective and produced a greater yield
than other patents and procedures. NMR, IR, mass spectroscopy, melting
point, and the positive optical rotation all verify that α-FMH was successfully
synthesized.
With future research into the modeling and docking study, we will be
able to predict binding activity and suggest reasonable active site residues. We
will also be able to suggest the residue involved in covalent bonding with α-
FMH. This active site will allow us to examine the potential for other possible
inhibitors using a virtual screening.
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Methods
The synthesis followed 3 chemical steps that are outlined in scheme 1.
These 3 steps comprise a novel and cost effective method for preparation of α-
FMH from aziridine 1 (6), scheme 1. The last step, 4 to 5, is an effort to isolate
the free amino acid without the di-hydrochloride salt.
1. 0.03 moles methyl 2- aziridinyl-3-(N-triphenylmethyl-4-imidazolyl)
propionate 1 (6) was fluorinated with 0.37 moles HF/Pyridine (70% : 30%)
in toluene at 70°C and stirring to fluorinate and remove the trityl group to
give crude 3. Note: 10 equivalents had the best yield, but it was later
determined that 5 equivalents is sufficient.
2. Calcium carbonate was added to the two phase system and vacuum
filtration was used to isolate calcium fluoride.
3. The crude reaction product, methyl ester 3, was hydrolyzed with 6 N HCl to
form α-FMH 4.
4. Crude α -FMH di-hydrochloride was purified by a cation exchange column
using 0.5-6.0 N HCL.
5. Fractions were collected and concentrated to dryness.
Scheme 1. Novel synthesis of α-FMH
Purification:
Filtrate collected from the 6.0 N HCl elution was re-chromatographed using HCl
concentrations over 0.5-6.0 N. The Pauly’s test was used to test for histidine.
Identificiation:
Melting point, FT-IR, carbon and proton NMR’s (run in DMSO-d6 and D2O),
optical roation, and mass spectroscopy (run at Boehringer-Ingelheim for the
free amino acid monomer of α-FMH) were used for identification.
Sequence alignment:
A BLASTP (1) database search was performed on the 662 amino acid sequence
of Homo sapiens HDC.
Homology model building:
Swiss-Model (11) had the best homology model (previously built) using DOPA
decarboxylase as a model after minimization when compared to ModBase (20),
3D-Jigsaw Comparative Modeling (3, 4, 9), ESyPred3D Web Server 1.0 (16), and
Geno3D (8).
Energy minimization and model refinement:
All models were optimized and refined using Summa Lab’s Protein Refinement
Server (24), and then submitted to the Swiss-Model server (2) for structural
quality checks and energy evaluations.
Inhibitor docking:
α-fluoromethylhistidine was docked into HDC using DockingServer (5).
Results and Discussion
The fractions from 2.0 N HCl yielded 3.3 g of α-FMH from the first
column and the fraction from 3.0 N HCl resulted in 0.7 grams of the dimer or
trimer as indicated by a lower Rf value than the monomer from 2.0 N HCl and
later confirmed with NMR. The product from 2.0 N HCl was further crystallized
with ether and ethanol to give 0.37 grams of crystals. The fraction retrieved
with 6.0 N HCl was rechromatographed on a second column. The fraction from
2.0 N HCl was 1.2 grams and 1.3 grams was retrieved from the fraction of 3.0 N
HCl. The fraction retrieved from 6.0 N HCl had a negative Pauly’s test.
A total of 4.5 g was retrieved (3.3 grams and 1.2 grams of α-FMH
crystals) giving a yield of 57%. Some material was lost during chromatography
and due to the harsh conditions of 100°C and pH 1. These crystals had a
melting point of 194-215°C compared to the literature melting point of 217-
219°C (15).
The optical rotation in H2O at 22°C and 546 nm was +0.09° compared to
a literature rotation of +17.3° in CF3COOH/H2O (15).
Figure 1. FT-IR spectrum of the product isolated with 2 N HCl from column 1
Figure 2 shows the NMR spectra of our crystals obtained from elution
with 2.0 N HCl in column 1 in DMSO-d6. The chemical shifts at 4.80 and 4.92
ppm demonstrate fluorine spin-spin coupling. Each are split into a doublet due
to the long range coupling with the methylene group adjacent to the imidazole
ring.
A B
Figure 2. A- 1H-NMR (DMSO-d6) of α-FMH and B- 13C-NMR (DMSO) of α-FMH
Mass spectroscopy of the free amino acid monomer was run at
Boehringer-Ingelheim and the observed mass was 188.0845, seen in figure 3,
which matches the true molecular weight of α-FMH.
Figure 3. Mass spectra of α-FMH, free amino acid monomer
Therefore, all tests identified the product as the desired α-FMH.
Process development:
Ring-opening of aziridine 1 with KF in DMF, with a catalyst
(tetrabutylammonium hexafluorophosphate 98%) and without a catalyst and
under various temperatures and times resulted in no reaction.
Docking study:
The inhibitor, α-FMH, was docked into the histidine decarboxylase from
Swiss-Model which had a dFire of -706.81 and a QMean of -78.1822.
It was hypothesized that the fluorine of the ligand would interact with a
serine residue in the active site, most likely serine 354, and that the phosphoryl
group would interact with lysine 305. The best docked binding modes agreed
with these predictions. As can be seen in table 1, the dockings numbered 1
and 2 do not show interactions of the phosphoryl group with the lysine
residue, but instead were exposed to the solvent.
Table 1. Docking Models
Serine 354 (pink) in figure 4 A is where the covalent bond is suspected
to form. As can be seen in figure 4 B, the fluorine of the ligand is 3.46 Å away
from the oxygen of the serine so it is within an appropriate distance for a
covalent bond to form.
A B
Figure 4. A – Histidine decarboxylase ribbon diagram (Pink = serine 354), B -
Docking of α-FMH-PLP into histidine decarboxylase active site (white =
phosphoryl, royal blue = α-FMH, green = fluorine, light blue = serine 354, 3.46
= distance between fluorine and serine)
Number Estimated Free
Energy of
Binding
(kcal/mol)
Total
Intermolecular
Energy
(kcal/mol)
Interactions Of
Interest
Distance (Å)
1 -2.50 -5.12 F-Ser 354 3.46
2 -1.54 -3.97 F-Ser 151
F-Ser 196
2.80
3.19
3 +1.03 -3.40 F-Ser 354
P-Lys 305
3.09
3.88
4 +2.48 -0.80 F-Ser 151
F-Ser 196
P-Lys 305
3.35
3.11
3.51
N
N
H
NH2
O
CH3
O
F
N
N
NH
O
O
CH3
N
N
O
O
CH3
F
NH2
HF/pyridine
(S)-alpha Fluoromethylhistidine x 2HCl
HCl aqueous
N
N
H
NH2
OHO
F
HCl aqueous
N
N
H
NH2
OHO
F
NH4OH
(S)-alpha Fluoromethylhistidine
1
2
3 4
5
Fluorination
Remove Trityl
Protecting Group
Ester Hydrolysis