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Synthesis and Circular Dichroism studies of certain Peptide Motifs
(Project
Report submitted by Bhismadev Chakrabarti to the Indian Academy of Sciences for work done in the period 20th May to 20th July
, 2000,at the Centre for Biochemical Technology, Delhi ,under the supervision of Prof .Samir K .Brahmachari.)
With
most of the genomes fully sequenced now, the focus of future biology will obviously be on the functional aspects of the genome.
Hence proteomics(see ref.4)is expected to assume a centrestage in the coming decade. Upcoming fields such as pharmacogenomics
and predictive medicines would rely heavily on proteomics.
My project focusses on synthesis and studies of certain peptide
motifs, and hence can be categorized under 'elementary proteomics.'
Protein function is integrally related to its
structure. Hence it is essential to make detailed structural studies of proteins in order to get an idea of the exact mode
of their function or develop strategies to enhance/reduce their activity. In spite of there being thousands of proteins with
innumerable functions, there are certain common structural motifs that go to make up this whole gamut .These structural motifs
include alpha-helix ,beta-pleated sheet ,beta-turns, etc.
Some peptide motifs have been identified using computational
tools (ref.1) .They are present in several microorganisms as conserved sequences.
It is of sufficient interest to study
these non-variant peptide motifs in detail using available biophysical and biochemical tools.
My project involves the
synthesis and Circular Dichroism (CD) studies of a few(two/three) of these peptides.
I have synthesized two peptides,
using conventional Fmoc (9-Fluorenylmethoxycarbonyl) chemistry based solid phase synthesis. The exact details are as follows:-
Fmoc Chemistry based Solid Phase Peptide Synthesis:an introduction
The Fmoc group is normally introduced in Schotten-Baumann manner using the rather
stable chloroformate FmocCl. Dipeptide and tripeptide formation often poses a problem when the Fmoc -amino acid is prepared
by the Schotten-Baumann route. Alternatively, Fmoc-amino acids may be prepared using FmocCl but without oligomer formation
by reaction with pertrimethylsilylamino acids and base in aprotic solvents.
The Fmoc group is very stable in acidic reagents
but is cleaved swiftly under certain basic conditions. Piperidine (20% in DMF) is the routine reagent but other systems, e.g.fluoride
ion in DMF, are also effective. Deprotection with piperidine takes only a matter of seconds in room temperature. The mechanism
of cleavage is E1cb, via the stabilised dibenzocyclopentadienide anion; the dibenzofulvene produced reacts with piperidine
giving an adduct as the coproduct. (ref.5)Peptide terminal amino groups, however, neither induce cleavage nor trap dibenzofulvene
to a significant extent. This deprotective procedure does not affect Z or Boc groups or indeed most other modern protecting
groups.
The controlled formation of a peptide bond by condensation of two amino acids requires activation of the carboxyl
group of one residue with the other .This is achieved by using so-called coupling reagents. These must satisfy the following
criteria:
1) The reagent should be generally utilizable.
2) It should be easy to handle.
3) It should work rapidly
and supply high yields.
4) Secondary reactions should not occur.
5) It should not cause racemization during synthesis.
Various routes have been adopted in order to achieve proper activation (of the carboxyl group )and coupling(see ref.4,pp25-41)
.In the method adopted in my experiments,I have made use of the carbodiimide reagents.The particular carbodiimide used in
my case was N,N'-diisopropylcarbodiimide.In this case,primary activating event is addition of the carboxy group to the carbodiimide
functionality to give an O-acylisourea,which is a potent acylating agent.O-acylisourea formation is rapid,leading to peptide
either by immediate aminolysis or via a symmetrical anhydride,with the simultaneous formation of dicyclohexylurea.This urea
is soluble enough for removal by thorough washing in solid phase synthesis.However,the extremely reactive nature of the O-acylisourea
makes it susceptible to many unwanted side-reactions,extensive racemization of the carboxy component or formation of N-acylurea
by intramolecular acyl transfer.To avoid such problems,coupling is performed in the presence of a suitable a-nucleophile.This
reacts with the O-acylisourea before side reactions can intervene.Consequently,an acylating agent of a lower potency is formed,but
it is more discriminating and does not lead to racemization or other side reactions.The a-nucleophile used mostly these days(and
also used by me in my syntheses) is 1-Hydroxybenzotriazole(HOBt).
Methods and Materials
Sequences
to be synthesised: (from N terminal to C terminal)
1) LPGKLADC (hereafter referred to as the first peptide)
(Leu-Pro-Gly-Lys-Leu-Ala-Asp-Cys)
2)NADFDGDQMAVH (hereafter referred to as the second peptide)
(Asn-Ala-Asp-Phe-Asp-Gly-Asp-Gln-Met-Ala-Val-His)
Protected
Amino Acids Used: (all supplied by Novabiochem Inc.)
Amino Acid Protected Form Used
Aspargine Fmoc-Asn(trt)-OH
Glutamine Fmoc-Gln(trt)-OH
Histidine Fmoc-His(trt)-OH
Valine Fmoc-Val-OH
Methionine Fmoc-Met-OH
Phenylalanine
Fmoc-Phe-OH
Cysteine Fmoc-Cys(Trt)-OH
Aspartic Acid Fmoc-Asp(OtBu)-OH
Alanine Fmoc -Ala-OH
Leucine Fmoc-Leu-OH
Lysine Fmoc-Lys(Boc)-OH
Glycine Fmoc-Gly-OH
Proline Fmoc-Pro-OH
Leucine Fmoc-Leu-OH
Resin used: 2-Chlorotrityl
Chloride Resin (supplied by Advanced Chemtech) (loading: 1.5mmol/g)
Attachment of first amino acid to the resin
The resin was first dried in vacuo .500mg of resin
was then weighed out and taken in the reaction flask of the automated peptide synthesizer (ACT90: Advanced Chemtech). It was
then left to swell for half an hour after adding ~10mL solvent (DCM in this case). Following this the flask was emptied and
flushed with Nitrogen gas. Calculated amounts of the first amino acid from the C terminal (protected form), DIPEA (N,N'-diisopropylethylamine)and
DCM (~15mL) were added to the flask. Using the ACT90 software, the instrument was programmed such that the flask was shaken
well for 40 minutes and then emptied and flushed thoroughly with Nitrogen gas. This was followed by washing the whole resin
with DCM 2 to 3 times. This first coupling procedure was repeated once again, to achieve maximum loading of the resin. A small
amount of resin was then taken out and deprotected manually (see Deprotection of the N-terminus of Amino Acid section later)
.The Kaiser Test was then performed to obtain indirect proof of the first amino acid being successfully attached to the resin
.On getting a positive Kaiser test (for manually deprotected peptide), the peptide was then given for deprotection of the
N-terminus.
Deprotection of the N-terminus of Amino Acid
A 20% piperidine solution was prepared in DMF .The dry peptide-resin complex was shaken for ~12 minutes,
twice. Approximately 3mL of this solution was used per mg of resin taken. Use of 20% piperidine solution streams from the
fact that Fmoc (9-Fluorenylmethoxycarbonyl) group is base labile group and it is easily removed under mildly basic conditions.
However, base treatment to remove Fmoc group has its own share of associated problems (for a detailed discussion, see Ref.2).
Following base treatment, the peptide-resin complex was then subjected to 4 washings with DMF(~10-12mL per washing) and two
washings with DCM. The washings were repeated until the colour of the pH paper changed from basic to neutral. Following this
the Kaiser Test was performed on a small amount of the peptide resin complex taken in an ignition tube.
Kaiser Test
The following solutions were prepared:
1) 5g ninhydrin in 100mL ethanol
2) 80g of liquefied phenol
in 20mL of ethanol
3) 2mL of a .001M aq. solution of potassium cyanide in 98mL pyridine
A few resin beads were
taken and washed several times with ethanol. They were then transferred to an ignition tube and 2 drops of solution 1, and
a drop each of the other two solutions were added. These were mixed well and kept in an oven (at around110C) for ~5minutes.
A positive test is indicated by blue resin beads, i.e. it suggests the presence of free primary amino groups.
End-capping
of remaining reactive trityl groups
End-capping refers to the process of blocking all the active trityl groups on the resin to which the first
amino acid has not attached itself. This is essential in order to prevent the formation of deleted sequences. This process
precedes the first deprotection procedure.
About 5mL HPLC grade methanol was added to the reaction flask and mixed for
15 minutes. Following this the resin was filtered and washed 3 times with DCM (~10mL/g of resin) twice with DMF, twice with
DCM and thrice with methanol, in that order. The resin was then dried in vacuo.
Elongation of the peptide chain
Calculated amounts of the amino acid (protected form), DIPCI (N,N'-diisopropylcarbodiimide), HOBt
(1-Hydroxybenzotriazole) and DCM (~10mL) were added to the flask.Using the ACT90 software, the instrument was programmed such
that the flask was shaken well for 40 minutes and then emptied and flushed thoroughly with Nitrogen gas. This was followed
by washing the whole resin with DCM 2 to 3 times. The Kaiser Test was then performed to obtain indirect proof of the amino
acid being successfully attached to the resin. On getting a negative Kaiser test (beads not changing colour), the peptide
was then deprotected to remove the Fmoc group.
Cleavage of completely deprotected peptide from resin
Before
cleavage the peptide-resin complex was washed 4-5 times each with methanol and cold ether and then dried thoroughly in a dessicator.
It was then transferred to a conical flask and then treated with a cleavage cocktail which was a variant of Reagent K (see
ref .3), keeping in mind the nature of the resin. The exact ratio of the individual components were as follows:
Ethanedithiol
(EDT): DCM: phenol: 30%TFA solution in DCM=5:5:5:85
The resin was shaken after every 5 minutes and this treatment was
continued for about 45 minutes.
Following this, the solution was filtered through a sintered funnel, and the filtrate
was concentrated using a Rotavapor (Buchi).
Purification of the peptide
a) Desalting
A
Sephadex G10 column was packed and the peptide was loaded into it after the column had been equilibrated with 20%acetic acid
solution (aqueous, degassed) for about an hour. Nearly 40 fractions were collected of ~3mL each. They were analyzed for presence
of peptide by spotting on the TLC plates followed by spraying with ninhydrin and heating for 5 minutes at around 100?The spots
which turned blue indicated the presence of peptide in those fractions. These fractions were collected in a flask and lyophilized
completely. Then this crude peptide was stored at -70?
b) Purification by HPLC
About 1mg of the crude
peptide was dissolved in 1mL of water and ~40??f the solution was loaded into the HPLC machine for an analytical run in the
isocratic mode (solvent system used was 30% acetonitrile in water with 0.05% TFA. HPLC was done in 2 machines (Waters and
Shimadzu) and the profile from both showed the presence of a major product (~70%) and a minor product (~20%). The major fraction
was collected by loading about 13mg of crude peptide dissolved in 1mL water into the Shimadzu machine and running the column
at a semi-preparative scale. This fraction was then lyophilized and given for mass spectrometry.
The second peptide when
analysed through the Shimadzu machine gave a profile corresponding to a 97% pure product.However, other characterisation studies
failed to prove the purity of this product.
c) Mass Spectrometry:
The mass chromatogram of
the first peptide showed that the mass of the major product was of mol. wt. 815.64. The calculated mass of the peptide is
815.9. Hence the purity of the product was proved.
However,the mass spectrum of the second peptide showed two major peaks
corresponding to molecular weights of 1561 and 1350,whereas the calculated mass of the peptide is 1319.Possible reason for
the peak corresponding to the mol.wt. of 1561 is that the final deprotection at the N-terminal did not occur properly, which
therefore led to isolation of the peptide with an N-terminal Fmoc group.(it is to be noted that the molecular weight of Fmoc
group is almost exactly equal to the difference between the peak corresponding to the higher mass and the calculated mass
of the peptide).While a plausible explanation could not be found for the second major peak (corresponding to a mass of 1350),conjectures
can certainly be made regarding the Methionine side chain (containing a -SCH3 group) undergoing certain unwanted reactions.
d) Sequencing
The first peptide was sequenced from the N terminus to the C terminus, using a Procise
Protein sequencer. The data showed the presence of all the seven amino acids in the expected order (Cysteine was not detected,
as it requires additional derivatization of the Cysteine moiety in order for it to be detected by the Sequencer.)
The
second peptide ,when analysed through the sequencer, gave very weak signals corresponding to the component amino acids, thereby
indicating a probable low yield of the desired product.
Circular Dichroism: A Primer
It is now possible to generate a wide variety of different proteins via solid-phase synthesis and recombinant
DNA methods. There is a need to determine the structure of larger and larger numbers of proteins. High resolution techniques
such as NMR and X-ray crystallography (that are complex and time-consuming) will be overwhelmed. Therefore, there is a need
for quick low resolution techniques for determining protein structure. For example, absorption, Raman, fluorescence, and circular
dichroism .
Plane polarized light can be assumed to be made of right and left circularly polarized light. When plane polarized
light passes through a solution of an optically active compound (in our case, compounds with chiral chromophores),the right
and left polarized components are absorbed to different extents. These components then recombine to give elliptically polarized
light with net ellipticity given by ?.
Electronic transitions like the pi-pi* and n-pi* give rise to characteristic absorbance
peaks when the molar ellipticity of the compound is plotted against the wavelength for a certain range of wavelengths (260nm
to 190nm for peptide chromophores).Based on the position, shape and nature of the peak/trough, qualitative (or, at best, semi-quantitative)estimates
can be made about the secondary structure of the peptide under study. However, conclusive evidence for exact structure can
come only from high resolution methods like nmr spectroscopy and x-ray crystallography. CD being a low resolution method ,has
the advantage of being quicker and less cumbersome.Hence it is more often used for monitoring conformational changes under
varying circumstances (e.g. monitoring conformational changes with changing pH,or changing temperature,etc.).TFE or tetrafluoroethanol,is
a solvent that has a remarkable property of stabilising a particular peptide conformation.Hence TFE studies(the CD is recorded
with varying concentrations of TFE) give us an idea of the actual conformation of the peptide.
CD Study
A CD study of the first purified peptide was done in a Jasco CD machine and the CD plot resembled that
of a random coil. A temperature study showed that the degree of randomness increased with increasing temperature. TFE studies
did not yield any significant results.
References
1)Dash Debasis,Brahmachari Samir K.:Unpublished results
2)Fields
et.al.:Variable success of peptide-resin cleavage and deprotection following solid phase peptide synthesis in Techniques in
Protein ChemistryV (J.W.Crabb,ed.),1994,pp501-507
3)King,D.S.,Fields ,C.G. and Fields,G.B.:Int.J.Pep.Prot.Res.,36,255(1990)
4)Brahmachari et.al. :"Multitude of 'omics' and 'omes';terms in Molecular Biology in the New Milennium",Current Science,September
1999
5)Jones,J.H. Amino Acid and Peptide Synthesis,Oxford University Press,1991
6)Technical Application Notes,Peptides
and Peptidomimetic Synthesis,Fluka Chemika 2000
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