Title of Invention

A PROCESS FOR PREPARATION OF A NOVEL SYNTHETIC PEPTIDE EPITOPE USEFUL FOR DIAGNOSIS OF ASPERGILLOSIS.

Abstract This invention relates to a A process for the preparation of a novel synthetic peptide epitope having the amino acid sequence isoleucyl-asparaginyl-glutaminyl-glutaminyl-leucyl-asparaginyl-prolyl-lysine (INQQLNPK) useful for the diagnosis of aspergillosis which comprises loading suitably protected isoleucine with appropriately functionalised polystyrene resin in presence of organic solvent, treating the said isoleucine loaded resin with deblocking agents such as herein defined, thereby deblocking the protecting moiety from the -amino group of isoleucine, coupling a suitably protected asparagines with the free -amino group of isoleucine obtained after deblocking the protecting moiety of said isoleucine using conventional coupling reagents, repeating the steps of coupling and deblocking sequentially with suitably protected glutamine, glutamine, leucine, asparagines, proline and lysine, drying the resin coupled with desired peptide sequence by conventional methods, cleaving of the said peptide from the resin by acid treatment, neutralizing the cleaved peptide with conventional methods, deblocking of the protecting groups of the side chains of the various amino acids, followed by hydrogenation and repeated precipitation to obtain the desired peptide.
Full Text The present invention relates to a process for the preparation of a novel synthetic peptide epitope having the amino acid sequence isoleucyl-asparaginyl-glutaminyl-glutaminyl-leucyl-asparaginyl-prolyl-lysine (INQQLNPK) useful for the diagnosis of Aspergillosis. The synthetic peptide epitope of the present invention has the following sequence: Isoleucyl-asparaginyl-glutaminyl-glutaminyl-leucyl-asparaginyl-prolyl-lysine (Ile-Asn-Gln-Gln-Leu-Asn-Pro-Lys) (INQQLNPK).
The above said peptide is useful for diagnosis of aspergillosis by enzyme linked immunosorbent assay. The peptide of the present invention can be used to raise polyclonal as well as monoclonal antibodies in mice which are useful for diagnosis of invasive aspergillosis by antigen detection.
Reference can be made to Clinical Microbiological reviews, Oct. 1991, p. 439-56, wherein fungi, Aspergillus fumigatus causes a wide spectrum of human and animal disorders such as allergic bronchopulmonary aspergillosis (ABPA), extrinsic allergic alveolitis, aspergilloma, and invasive aspergillosis. Invasive form of aspergillosis is becoming increasingly important in immunosuppressed conditions due to environmental pollution, enhanced use of chemotherapeutic drugs and antibiotics etc. The most susceptible hosts are the immunocompromised patients, such as the cases with organ transplant, leukemia, or acquired immunodeficiency syndrome (AIDS). Currently available tests for identification of this fungi is based on tedious, time consuming , less sensitive methods such as microscopy, cultures, electrophoresis and immunodiffusion. Many clinical features of aspergillosis are similar to tuberculosis and most of the

aspergillosis patients are put on antituberculous therapy. The microscopy of the specimen for identification of Aspergillus hyphae is not easy under field conditions and specimen from ihc patients in the early stages of disease are often negative in the direct mounts. Further, the fungal culture generally takes 3-4 weeks and is expensive as a routine diagnostic measure. The widely used skin testing for Aspergillus allergic patients lacks sensitivity and specificity as the mixture of allergens used for testing is not well characterised and needs standardisation. The steroid therapy used for allergic patients and chemotherapy for invasive patients are more beneficial when employed in the early stages of the disease. Consequent to these factors, many investigators recommend that early diagnosis of aspergillosis should be considered as a priority area of research and development.
Reference can be made to J. Med. Vet. Mycol. 22 : 381-394, Int. Arch. Allergy Appl. Immunol. 70: 108-111 wherein an enzyme linked immunosorbent assay (ELISA). based sensitive test using a mixture of potent Aspergillus fumigatus antigens has been developed which provides a rapid diagnosis in early stages of the disease. However, there are variations in the mixture obtained from various clinical isolates of Aspergillus fumigatus. Further, the present test is antibody based and thus is not useful for invasive patients.
Reference can be made to Mycopathologia, 106 : 121-129, 1989, Applied Environmental Microbiology, 56: 2441-2452, 1990, Journal of Genetic Microbiology, 136: 1991-1994, 1990, Journal of Clinical Microbiology, 29 : 2099-2106, 1991,wherein an alternative method of diagnosis of aspergillosis has been the use of polymerase chain reaction (PCR)

and hybridisation. Various groups have identified and synthesised primers for genes
specific to A. fumigatus.
Reference can be made to Preparative biochemistry, 25 : 171-181, 1995, wherein a PCR
based test specific for A. fumigatus has been developed. Gene based tests are highly
sensitive and specific in comparison to immunodiagnosis. However, at present no gene
based diagnostic kit for aspergillosis is available in the market as routine use of this test
proves very expensive.
Reference can be made to Methods in Enzymology, 178 : 571-717, wherein, peptides
mimicking the epitope in native antigens have been utilised in diagnosis as well as
therapy of hepatitis, influenza, malaria, AIDS etc. Peptides based diagnosis of
aspergillosis would be standardised and cost-effective. Hence, an antigenic determinant
of a major allergen/ antigen (Asp f 1) secreted by Aspergillus fumigatus was identified
and synthesised. Asp f 1 is diagnostically relevant for allergic as well as invasive
aspergillosis.
Reference can be made to J. Immunology, 149: 454-60, 1992, J.Immunology 149: 3354-
3359, 1992, XVI IUBMB Abstract S-142, 1994 wherein the gene for Asp f 1 has been
cloned and sequenced. The three genes were isolated from different strains however, the
sequences obtained from all the three were similar.
Reference can be made to Peptides, 17: 183-190, 1996, wherein some peptides of Asp f 1
have been synthesised but none of the sequences revealed by them is related to the
sequence of the present invention. Further, they have not indicated diagnostic relevance
of these peptides.

The present synthetic peptide is a sequential epitope which is able to bind the A. fumigatus specific IgG and IgE antibodies in the sera of patients of aspergillosis. Further, it is able to raise significant amount of antibodies in the mit.^ and these polyclonal antibodies can be used for diagnosis of invasive aspergillosis.
The main object of the present invention is to provide a process for the preparation of a novel synthetic peptide epitope useful for the diagnosis of aspergillosis which obviates the drawbacks as detailed above.
Accordingly the present invention provides a process for the preparation of a novel synthetic peptide epitope having the amino acid sequence isoleucyl-asparaginyl-glutaminyl-glutaminyl-leucyl-asparaginyl-prolyl-lysine (INQQLNPK) useful for the diagnosis of aspergillosis which comprises loading suitably protected isoleucine with appropriately functionalised polystyrene resin in presence of organic solvent , treating the said isoleucine loaded resin with deblocking agents such as herein defined , thereby deblocking the protecting moiety from the oc -amino group of isoleucine , coupling a suitably protected asparagines with the free oc -amino group of isoleucine obtained after deblocking the protecting moiety of said isoleucine using conventional coupling reagents, repeating the steps of coupling and deblocking sequentially with suitably protected glutamine, glutamine, leucine, asparagine, proline and lysine, drying the resin coupled with desired peptide sequence by conventional methods, cleaving of the said peptide from the resin by acid treatment , neutralising the cleaved peptide with conventional methods, deblocking of the protecting groups of the side chains of the various amino acids, followed by hydrogenation and repeated precipitation to obtain the desired peptide.

The polystyrene resin used may be such as Merrifield's resin, PAM resin, Wang resin etc. The groups for protecting ot-amino and side chain functions of amino acids used may be such as t-butyloxycarbonyl (Boc), benzoyloxycarbonyl (Z), 2-chlorobenzoylcarbonyl (Cl-Z), 9-fluorenylmethoxycarbonyl (FMOC) and benzyl, methyl esters respectively. The attachment of suitably protected isoleucine with the resin may be carried out using ester or amide linkage with suitable reagents such as dicyclohexylcarbodiimide (DCC)/ Dimethyl amino pyridine (DMAP), cesium salt or using a linker (for PAM resin) by DCC alone. The coupling reaction was performed in organic solvents such as methylene chloride, dicholoromethane, dimethylformamide, ether, petroleum ether, acetic acid, methanol, N-methyl-2-pyrolidone etc.
The deblocking agents used for the removal of a-amino protecting group may be such as hydrochloric acid (HC1)/ dioxane, trifluoroacetic acid (TFA)/ CHaCh, HBr/ Acetic acid (AcOH), formic acid etc. For deblocking L-tyrosine thioanisole has been used as a scavenger.
The coupling during solid phase synthesis was carried out by using agents such as dicyclohexylcarbodiimide (DCC), mixed anhydride, active esters, Benzotriazole-1-yl-oxi-tris (dimethyl amino)- phosphonium hexafluorophosphate (BOP reagent) and 2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) etc.

Cleavage of the peptide from the resin may be carried out using the acids such as HBr/
AcOH, TFA/ Trifluoro methane sulfonic acid (TFMSA), liquid ammonia, liquid HF or
trifluoro acetic acid etc.
The neutralisation may be carried out by tertiary bases such as N-methylmorpholine,
troethyl amine or diisopropylethyl amine.
The hydrogenation agents used maybe such as Pd/ C, Palladium chloride, Rhodium/ C,
adams catalysts and palladium black etc. The precipitation may be carried out using
anhydrous methanol and dry ether.
The present invention describes a novel epitope of Asp f 1, a major allergen/ antigen, of
A. fumigatus. As mentioned in the prior art, a few peptides of Asp f 1 have been reported
but their relevance for diagnosis has not been shown. Thus, a novel diagnostically
relevant antigenic determinant has been synthesised with potential applications in
immunotherapy and immunoprophylaxis of aspergillosis.
The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of the present invention.
Example 1
Fmoc-protected L-Isoleucine was esterified with 1.0 g p-alkoxybenzyl alcohol functionalised polystyrene resin (Wang resin) having degree of substitution 0.72 millimoles per gram of the solid support. After the esterification the protected threonine was loaded on the resin and found to be 0.65 millimoles per gram of the resin.

The above resin 1.0 g was placed in a manual synthesis vessel and treated with 20% piperidine in dichloromethane (DCM) for 1.5 minutes and dimethyl formamide (DMF) for 20 minutes respectivel> under stirring with nitrogen cur, • :t. Then the resin was washed with dichloromethane and mixture of dichloromethane and dimethylformamide with a ratio of 1:1. The free a-amino group of isoleucine, thus generated was acylated with three molar excess of suitably protected asparagine (2 mM) using 1-hydroxybenzotriazole and diisopropyl carbodiimide both in three molar excess in DMF/ DCM mixture (1: 1) for a period of two hours. The completion of reaction was monitored by Kaiser's test. After complete coupling the resin was washed with a mixture of dichloromethane and dimethylformamide with a ratio of 1:1. The sequence of deblocking and coupling was carried in the same manner as described above with suitably protected Gin, Gin, Leu, Asn, Pro and Lys respectively. The resin was then removed from the vessel and dried over P2Os under vacuum. The dried resin (1.8 g) was treated with 95% trifluoroacetic acid and 5% thioanisol mixture (45 ml) for a period of 1.5 hrs. The contents were filtered and the filtrate was concentrated to half the volume. Addition of dry ether resulted in the precipitation of white hygroscopic powder. The crude product was precipitated with anhydrous methanol and ether to get the desired undecapeptide. The crude material was purified by reverse phase HPLC. All the reactions were carried out at 25°C. Yield: 0.38g, 42.5%,
Fast atom bombardment mass spectrum (FABMS) : [M+H+]+, 954,
Homogeneous on thin layer chromatography (TLC) and High performance liquid chromatography (HPLC).

Example 2
Boc-protected L- Isoleucine was esterified with 0.75 g functionalised polystyrene resin (Merrifield) having degree of substitution 0.9 millimoles per gram of the solid support. After the esterification the protected isoleucine was loaded on the resin and found to be 0.51 millimoles per gram of the resin.
The above resin 820 mg was placed in a manual synthesis vessel and treated with 50% TFA in dichloromethane for 2 minutes and dimethyl formamide (DMF) for 20 minutes respectively under stirring with nitrogen current. Then the resin was washed with dichloromethane and mixture of dichloromethane and dimethylformamide with a ratio of 1:1 and subsequently washed three times with 20% diisopropyl ethylamine (DIEA) in dichloromethane and finally three times with dichloromethane.
The free a-ammo group of isoleucine, thus generated was acylated with three molar excess of suitably protected asparagine (1.6 mM) using 1-hydroxybenzotriazole, DIEA and BOP reagent all in three molar excess in DMF/ DCM mixture (1: 1) for one hour. The completion of reaction was monitored by Kaiser's test. After complete coupling the resin was washed with a mixture of dichloromethane and dimethylformamide with a ratio of 1:1. The sequence of deblocking and coupling was carried in the same manner as described above with suitably protected Gin, Gin, Leu, Asn, Pro and Lys respectively. The resin was then removed from the vessel and dried over P2O5 under vacuum. The dried resin (1.93 g) was treated with trifluoroacetic acid and trifluoromethanesulfonic acid (9: 1) and 5% thioanisol mixture (20 ml) for a period of 1.5 hrs. The contents were filtered and the filtrate was concentrated to half the volume. Addition of dry ether resulted in the precipitation of white hygroscopic powder. The crude product was

precipitated with anhydrous methanol and ether to get the desired undecapeptide. The
crude material was purified by reverse phase HPLC. All the reactions were carried out at
25°C.
Yield: 0.18g, 28%,
Fast atom bombardment mass spectrum (FABMS) : [M+H+]+, 954,
Homogeneous on thin layer chromatography (TLC) and High performance liquid
chromatography (HPLC).
Example 3
Boc-protected L-Isoleucine was esterified with 0.75 g functionalised polystyrene resin (Merrifield) having degree of substitution 0.9 millimoles per gram of the solid support. After the esterification the protected threonine was loaded on the resin and found to be 0.52 millimoles per gram of the resin.
The above resin 870 mg was placed in a manual synthesis vessel and treated with 50% TFA in dichloromethane (DCM) for 2 minutes and dimethyl formamide (DMF) for 20 minutes respectively under stirring with nitrogen current. Then the resin was washed with dichloromethane and mixture of dichloromethane and dimethylformamide with a ratio of 1:1 and subsequently washed with 20% DIEA in dichloromethane (three times) and finally with dichloromethane (three times).
The free a-amino group of isoleucine, thus generated was acylated with three molar excess of suitably protected asparagine pentafluorophenyl ester (1.6 mM) in DMF/DCM mixture (1:1) for a period of two and a half hours. The completion of reaction was monitored by Kaiser's test. After complete coupling the resin was washed with a mixture of dichloromethane and dimethylformamide with a ratio of 1:1. The sequence of

deblocking and coupling was carried in the same manner as described above with suitably protected Gln, Gln, Leu, Asn, Pro and Lys respectively. The resin was then removed from the vessel and dried over P2O5 under vacuum. The dried resin (2.0 g) was treated with trifluoroacetic acid and trifluoromethanesulfonic acid (9: 1) and 5% thioanisol mixture (20 ml) for a period of 1.5 hrs. The contents were filtered and the filtrate was concentrated to half the volume. Addition of dry ether resulted in the precipitation of white hygroscopic powder. The crude product was precipitated with anhydrous methanol and ether to get the desired undecapeptide. The crude material was purified by reverse phase HPLC. All the reactions were carried out at 25°C. Yield: 0.24g, 35%,
Fast atom bombardment mass spectrum (FABMS) : [M+H+]+, 954,
Homogeneous on thin layer chromatography (TLC) and High performance liquid chromatography (HPLC).
Disclosed is a process for preparation of a novel synthetic peptide epitope useful for the diagnosis of aspergillosis with a sequence isoleucyl-asparaginyl-glutaminyl-glutaminyl-leucyl-asparaginyl-prolyl-lysine (INQQLNPK) . The peptide was synthesised by solid phase synthesis. The peptide was able to bind A. fumigatus specific antibodies in the sera of patients and hence, useful in enzyme linked immunosorbent assay (ELISA) for the diagnosis of aspergillosis.
The main advantages of the present invention are :

1. The synthetic peptide epitope of the present invention would facilitate use of pure,
homogeneous, cost-effective, specific diagnostic reagent for aspergillosis. Such a
reagent is anticipated to find a place in international market for diagnostics for
universal application.
2. Simple and rapid diagnostic technologies for aspergillosis can easily be developed
with the present peptide.
3. An immunodiagnostic kit based on the polyclonal or monoclonal antibodies raised
against this antigenic determinant could also be used for detection of antigen in
patients of invasive aspergillosis.





WE CLAIM
1. A process for the preparation of a novel synthetic peptide epitope having the amino
acid sequence isoleucyl-asparaginyl-glutaminyl-glutaminyl-leucyl-asparaginyl-
prolyl-lysine (INQQLNPK) useful for the diagnosis of aspergillosis which comprises
loading suitably protected isoleucine with appropriately functionalised polystyrene
resin in presence of organic solvent , treating the said isoleucine loaded resin with
deblocking agents such as herein defined , thereby deblocking the protecting moiety
from the oc -amino group of isoleucine , coupling a suitably protected asparagines
with the free oc -amino group of isoleucine obtained after deblocking the protecting
moiety of said isoleucine using conventional coupling reagents, repeating the steps
of coupling and deblocking sequentially with suitably protected glutamine,
glutamine, leucine, asparagine, proline and lysine, drying the resin coupled with
desired peptide sequence by conventional methods, cleaving of the said peptide
from the resin by acid treatment, neutralising the cleaved peptide with conventional
methods, deblocking of the protecting groups of the side chains of the various amino
acids, followed by hydrogenation and repeated precipitation to obtain the desired
peptide.
2. A process as claimed in claim 1 wherein the polystyrene resin used is selected from
Merrifield' resin, PAM resin, Wang resin .
3. A process as claimed in claim 1 & 2 wherein the loading of suitably protected
isoleucine with the resin may be carried out using ester or amide linkage.

4. A process as claimed in claim 1 to 3 wherein the organic solvents used is selected
from methylene chloride, dichloromethane, dimethylformamide, ether, petroleum
ether, acetic acid, methanol N-methyl-2-pyrolidone .
5. A process as claimed in claim 1 to 4 wherein the deblocking agents used is selected
from HCl/dioxane, TFA/ CH2C12, HBr/AcOH, formic acid .
6. A process as claimed in claim 1 to 5 wherein the coupling is carried out by using
agents such as dicyclohexylcarbodiimide, mixed anhydride, active esters, BOP
reagent, HBTU, triethylamine and ethylchloroformate.
7. A process as claimed in claim 1 to 6 wherein cleavage of the peptide from the resin is
carried out using the acids such as HBr/AcOH, TFA/TFMSA, liquid ammonia, liquid
HF and trifluoroacetic acid etc.
8. A process as claimed in claim 1 to 7 wherein the neutralisation is carried out by
tertiary bases such as N-methylmorpholine, triethyl amine or diisopropylethyl amine.
9. A process as claimed in claim 1 to 8 wherein the hydrogenation agents used is
selected from Pd/C, Palladium chloride, Rhodium/C, adams catalysts and palladium
black.

10. A process for the preparation of novel synthetic peptide epitope having the amino acid sequence isoleucyl-asparaginyl-glutarninyl-glutaminyl-leucyl-asparaginyl-prolyl-lysine (INQQLNPK) useful for the diagnosis of aspergillosis substantially as herein described with reference to the examples.

Documents:

751-del-1998-abstract.pdf

751-del-1998-claims.pdf

751-del-1998-complete specification (granted).pdf

751-del-1998-correspondence-others.pdf

751-del-1998-correspondence-po.pdf

751-del-1998-description (complete).pdf

751-del-1998-form-1.pdf

751-del-1998-form-2.pdf


Patent Number 188957
Indian Patent Application Number 751/DEL/1998
PG Journal Number 48/2002
Publication Date 30-Nov-2002
Grant Date 19-Sep-2003
Date of Filing 24-Mar-1998
Name of Patentee COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH
Applicant Address RAFI MARG,NEW DELHI-110001,INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 PRIYANKA PRIYADARSINY BIOCHEMICAL TECHNOLOGY,MALL ROAD,NEW DELHI-7,INDIA.
2 PURANAM USHA SARMA BIOCHEMICAL TECHNOLOGY,MALL ROAD,NEW DELHI-7,INDIA.
3 TARUNA MADAN BIOCHEMICAL TECHNOLOGY,MALL ROAD,NEW DELHI-7,INDIA.
4 SETURAM BANDACHARYA KATTI CENTRAL DRUG RESEARCH INSTITUTE,LUCKNOW-226001,INDIA.
5 WAHAJUL HAG CENTRAL DRUG RESEARCH INSTITUTE,LUCKNOW-226001,INDIA.
PCT International Classification Number C07K 1/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA