US20080199483A1

US20080199483A1 - Long lasting fusion peptide inhibitors of viral infection

Info

Publication number: US20080199483A1
Application number: US11/877,221
Authority: US
Inventors: Dominique P. Bridon; Robert S. Dufresne; Nissab Boudjellab; Martin Robitaille; Peter G. Milner
Original assignee: ConjuChem Biotechnologies Inc
Current assignee: Conjuchem LLC
Priority date: 1999-05-17
Filing date: 2007-10-23
Publication date: 2008-08-21
Also published as: EP2110381A1; US20050070475A1; US7608271B2; WO2000069902A1; EP1179012B2; ES2185595T3; ATE226593T1; ATE443714T1; BR0010757A; CA2372338A1; JP2002544287A; AU761591B2; EP1179012B1; US7582301B1; ES2185595T5; HK1053128A1; DE60000665T3; DK1264840T3; PT1264840E; JP2009167208A

Abstract

Peptides exhibiting anti-viral and anti-fusogenic activity are modified to provide greater stability and improved half-life in vivo. The selected peptides include fusion inhibitors DP178 and DP107 and related peptides and analogs thereof. The modified peptides are capable of forming covalent bonds with one or more blood components, preferably a mobile blood component.

Description

FIELD OF THE INVENTION

This invention relates to modified peptides that are inhibitors of viral activity and/or exhibit antifusogenic properties. In particular, this invention relates to modified peptide inhibitors of human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), human parainfluenza virus (HPV), measles virus (MeV), and simian immunodeficiency virus (SIV) with long duration of action for the treatment of the respective viral infections. The invention also relates to conjugates of the modified peptides and endogenous carriers, particularly conjugates of the modified peptides and various mobile blood components, particularly mobile endogenous proteins.

BACKGROUND OF THE INVENTION

Membrane fusion events, while commonplace in normal cell biological processes, are also involved in a variety of disease states, including, for example the entry of enveloped viruses into cells. Peptides are known that inhibit or otherwise disrupt membrane fusion-associated events, including, for example, inhibiting retroviral transmission to uninfected cells. As an example, the synthetic peptides DP-107 and DP-178 derived from separate domains within the human immunodeficiency virus type 1 (“HIV-1”) transmembrane (“TM”) glycoprotein gp41, are potent inhibitors of HIV-1 infection and HIV induced cell-cell fusion.
Lambert, et al., “Peptides from Conserved Regions of Paramyxovirus Fusion (F) Proteins are Potent Inhibitors of Viral Fusion,” Proc. Natl. Acad. Science U.S.A., Mar. 5, 1996, Vol. 93 (5), pp. 2186-91, discloses that the synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp41, are potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), Lambert, et al. identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV) blocked homologous virus-mediated syncytium formation and exhibited EC₅₀values in the range 0.015-0.250 μM. Moreover, these peptides were highly selective for the virus of origin.
U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 incorporated herein in their entirety, likewise disclose that the 36 amino acid peptide DP178 corresponding to amino acids 638 to 673 of gp41 from the HIV-1 isolate LAI (HIV-1_LAI), and the 38 amino acid peptide DP107 corresponding to amino acids 558-595 of gp41 from the HIV-1_LAI, both exhibit potent anti-HIV-1 activity.
While many of the anti-viral or anti-fusogenic peptides described in the art exhibit potent anti-viral and/or anti-fusogenic activity, these peptides suffer from short plasma half-lifes in vivo, primarily due to rapid serum clearance and peptidase and protease activity. This in turn greatly reduces the effective anti-viral activity of the peptides. There is therefore a need for a method of prolonging the half-life of existing anti-viral and/or anti-fusogenic peptides and providing for longer duration of action of these peptides in vivo.

SUMMARY OF THE INVENTION

The present invention meets these and other needs and is directed to modified peptides having anti-viral activity and/or anti-fusogenic activity. These modified peptides provide for an increased stability in vivo and a reduced susceptibility to peptidase or protease degradation. These modified peptides thereby minimize, e.g., the need for more frequent, or even continual, administration of the peptides. The products of varying embodiments of the present invention can be used, e.g., as a prophylactic against and/or treatment for infection of a number of viruses, including human immunodeficiency virus (HIV), human respiratory syncytial virus (RSV), human parainfluenza virus (HPV), measles virus (MeV) and simian immunodeficiency virus (SIV). Modification of other peptides involved in viral transfection (e.g., Hepatitis, Epstein Barr and other related viruses) is also within the scope of the invention.
This invention relates to chemically reactive modifications of peptides exhibiting anti-viral and/or anti-fusogenic activity such that the modified peptides can react with available functionalities on blood components to form stable covalent bonds. In one embodiment of the invention, the modified peptides comprise a reactive group which is reactive with amino groups, hydroxyl groups, or thiol groups on blood components to form stable covalent bonds. In another embodiment of the invention, the reactive group can be a maleimide which is reactive with a thiol group on a blood protein, including a mobile blood protein such as albumin.
In particular, the invention relates to such chemically reactive modifications of DP107 and DP178 peptides and analogs thereof, including peptides comprised of amino acid sequences from other (non-HIV) viruses that correspond to the gp41 region of HIV from which DP107 and DP178 are derived and that exhibit anti-viral or anti-fusogenic activity. More particularly, these peptides can exhibit anti-viral activity against, among others, human respiratory syncytial virus (RSV), human parainfluenza virus (HPV), measles virus (MeV) and simian immunodeficiency virus (SIV). The invention also relates to such chemically reactive modifications of the peptides of SEQ ID NO:1 to SEQ ID NO:86.
The invention also relates to compositions for use in the prevention and/or treatment of viral infection comprising a peptide that exhibits anti-viral activity modified with a reactive group as described. More particularly, the invention relates to such compositions for use in the prevention and/or treatment of AIDS, human respiratory syncytial virus (RSV), human parainfluenza virus (HPV), measles virus (MeV) and simian immunodeficiency virus (SIV).

BRIEF DESCRIPTION OF THE TABLES

The invention will be better understood by reference to the Tables, in which:
Table 1 lists the commonly occurring amino acids together with their one letter and three letter abbreviations, and common protecting groups.
Table 2 shows DP178 carboxy truncations.
Table 3 shows DP178 amino truncations.
Table 4 shows DP107 carboxy truncations.
Table 5 shows DP107 amino truncations.
Table 6 shows HIV-2_NIHZDP178 analog carboxy truncations.
Table 7 shows HIV-2_NIHZDP178 analog amino truncations.
Table 8 shows RSV F2 region DP107 analog carboxy truncations.
Table 9 shows RSV F2 region DP107 analog amino truncations.
Table 10 shows RSV F1 region DP178 analog carboxy truncations.
Table 11 shows RSV F1 region DP178 analog amino truncations.
Table 12 shows HPV3 F1 region DP 178 analog carboxy truncations.
Table 13 shows HPV3 F1 region DP 178 analog amino truncations.
Table 14 shows HPV3 F1 region DP 107 analog carboxy truncations.
Table 15 shows HPV3 F1 region DP 107 analog amino truncations.
Table 16 shows representative anti-RSV peptides.
Table 17 shows representative anti-HPV3 peptides.
Table 18 shows representative anti-SIV peptides.
Table 19 shows representative anti-MeV peptides.

BRIEF DESCRIPTION OF SEQUENCE LISTING

The invention will be better understood by reference to the Sequence Listing, in which:
SEQ ID NO:1 shows the peptide sequence of DP 178.
SEQ ID NO:2 shows the peptide sequence of DP 107
SEQ ID NO:3-9 show peptide sequences of certain DP178 analogs.
SEQ ID NO:10-30 show the peptide sequences of RSV F1 region and F2 region corresponding to DP178 and DP107, and representative anti-RSV peptides;
SEQ ID NO:31-62 show the peptide sequences of HPIV3 F1 region corresponding to DP178 and DP107, and representative anti-HPIV3 peptides;
SEQ ID NO:63-73 show peptide sequences of SIV corresponding to DP178 and representative anti-SIV peptides; and
SEQ ID NO:74-78 show peptide sequences of MeV corresponding to DP178 and representative anti-MeV peptides.

DETAILED DESCRIPTION OF THE INVENTION

To ensure a complete understanding of the invention the following definitions are provided:
Anti-viral peptides: As used herein, anti-viral peptides shall refer to peptides that inhibit viral infection of cells, by, for example, inhibiting cell-cell fusion or free virus infection. The route of infection may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving viral and cellular structures. Peptides that inhibit viral infection by a particular virus may be referenced with respect to that particular virus, e.g., anti-HIV peptide, anti-RSV peptide, etc.
Antifusogenic peptides: Antifusogenic peptides are peptides demonstrating an ability to inhibit or reduce the level of membrane fusion events between two or more entities, e.g., virus-cell or cell-cell, relative to the level of membrane fusion that occurs in the absence of the peptide.
HIV and anti-HIV peptides: The human immunodeficiency virus (HIV), which is responsible for acquired immune deficiency syndrome (AIDS), is a member of the lentivirus family of retroviruses. There are two prevalent types of HIV, HIV-1 and HIV-2, with various strain of each having been identified. HIV targets CD-4+ cells, and viral entry depends on binding of the HIV protein gp41 to CD4+ cell surface receptors. Anti-HIV peptides refer to peptides that exhibit anti-viral activity against HIV, including inhibiting CD-4+ cell infection by free virus and/or inhibiting HIV-induced syncytia formation between infected and uninfected CD-4+ cells.
SIV and anti-SIV peptides: Simian immunodeficiency viruses (SIV) are lentiviruses that cause acquired immunodeficiency syndrome (AIDS)-like illnesses in susceptible monkeys. Anti-SIV peptides are peptides that exhibit anti-viral activity against SIV, including inhibiting of infection of cells by the SIV virus and inhibiting syncytia formation between infected and uninfected cells.
RSV and anti-RSV peptides: Respiratory syncytial virus (RSV) is a respiratory pathogen, especially dangerous in infants and small children where it can cause bronchiolitis (inflammation of the small air passages) and pneumonia. RSVs are negative sense, single stranded RNA viruses and are members of the Paramyxoviridae family of viruses. The route of infection of RSV is typically through the mucous membranes by the respiratory tract, i.e., nose, throat, windpipe and bronchi and bronchioles. Anti-RSV peptides are peptides that exhibit anti-viral activity against RSV, including inhibiting mucous membrane cell infection by free RSV virus and syncytia formation between infection and uninfected cells.
HPV and anti-HPV peptides: Human parainfluenza virus (HPIV or HPV), like RSV, is another leading cause of respiratory tract disease, and like RSVs, are negative sense, single stranded RNA viruses that are members of the Paramyxoviridae family of viruses. There are four recognized serotypes of HPIV—HPIV-1, HPIV-2, HPIV-3 and HPIV-4. HPIV-1 is the leading cause of croup in children, and both HPIV-1 and HPIV-2 cause upper and lower respiratory tract illnesses. HPIV-3 is more often associated with bronchiolitis and pneumonia. Anti-HPV peptides are peptides that exhibit anti-viral activity against HPV, including inhibiting infection by free HPV virus and syncytia formation between infected and uninfected cells.
MeV and anti-Mev peptides: Measles virus (VM or MeV) is an enveloped negative, single-stranded RNA virus belonging to the Paramyxoviridae family of viruses. Like RSV and HPV, MeV causes respiratory disease, and also produces an immuno-suppression responsible for additional, opportunistic infections. In some cases, MeV can establish infection of the brain leading to severe neurlogical complications. Anti-MeV peptides are peptides that exhibit anti-viral activity against MeV, including inhibiting infection by free MeV virus and syncytia formation between infected and uninfected cells.
DP-178 and DP178 analogs: Unless otherwise indicated explicitly or by context, DP-178 means the 36 amino acid DP-178 peptide corresponding to amino acid residues 638-673 of the gp41 glycoprotein of HIV-1 isolate LAI (HIV_LAI) and having the sequence:
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 1)

as well as truncations, deletions and/or insertions thereof. Truncations of the DP178 peptide may comprise peptides of between 3-36 amino acids. Deletions consist of the removal of one or more amino acid residues from the DP178 peptide, and may involve the removal of a single contiguous portion of the peptide sequence or multiple portions. Insertions may comprise single amino acid residues or stretches of residues and may be made at the carboxy or amino terminal end of the DP178 peptide or at a position internal to the peptide.
DP178 peptide analogs are peptides whose amino acid sequences are comprised of the amino acid sequences of peptide regions of viruses other than HIV-1_LAIthat correspond to the gp41 region from which DP178 was derived, as well as an truncations, deletions or insertions thereof. Such other viruses may include, but are not limited to, other HIV isolates such as HIV-2_NIHZ, respiratory syncytial virus (RSV), human parainfluenza virus (HPV), simian immunodeficiency virus (SIV), and measles virus (MeV). DP178 analogs also refer to those peptide sequences identified or recognized by the ALLMOTI5, 107×178×4 and PLZIP search motifs described in U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 and incorporated herein, having structural and/or amino acid motif similarity to DP178. DP178 analogs further refer to peptides described as “DP178-like” as that term is defined in U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459.
DP-107 and DP107 analogs: Unless otherwise indicated explicitly or by context, DP-107 means the 38 amino acid DP-107 peptide corresponding to amino acid residues 558-595 of the gp41 protein of HIV-1 isolate LAI (HIV_LAI) and having the sequence:
(SEQ ID NO: 2)

NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

as well as truncations, deletions and/or insertions thereof. Truncations of the DP107 peptide may comprise peptides of between 3-38 amino acids. Deletions consist of the removal of one or more amino acid residues from the DP107 peptide, and may involve the removal of a single contiguous portion of the peptide sequence or multiple portions. Insertions may comprise single amino acid residues or stretches of residues and may be made at the carboxy or amino terminal end of the DP107 peptide or at a position internal to the peptide.
DP107 peptide analogs are peptides whose amino acid sequences are comprised of the amino acid sequences of peptide regions of viruses other than HIV-1_LAIthat correspond to the gp41 region from which DP107 was derived, as well as truncations, deletions and/or insertions thereof. Such other viruses may include, but are not limited to, other HIV isolates such as HIV-2_NIHZ, respiratory syncytial virus (RSV), human parainfluenza virus (HPV), simian immunodeficiency virus (SIV), and measles virus (MeV). DP107 analogs also refer to those peptide sequences identified or recognized by the ALLMOTI5, 107×178×4 and PLZIP search motifs described in U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 and incorporated herein, having structural and/or amino acid motif similarity to DP107. DP107 analogs further refer to peptides described as “DP107-like” as that term is defined in U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459.
Reactive Groups: Reactive groups are chemical groups capable of forming a covalent bond. Such reactive groups are coupled or bonded to a DP-107 or DP-178 peptide or analogs thereof or other anti-viral or anti-fusogenic peptide of interest. Reactive groups will generally be stable in an aqueous environment and will usually be carboxy, phosphoryl, or convenient acyl group, either as an ester or a mixed anhydride, or an imidate, thereby capable of forming a covalent bond with functionalities such as an amino group, a hydroxy or a thiol at the target site on mobile blood components. For the most part, the esters will involve phenolic compounds, or be thiol esters, alkyl esters, phosphate esters, or the like.
Functionalities: Functionalities are groups on blood components to which reactive groups on modified anti-viral peptides react to form covalent bonds. Functionalities include hydroxyl groups for bonding to ester reactive entities; thiol groups for bonding to maleimides, imidates and thioester groups; amino groups for bonding to carboxy, phosphoryl or acyl groups and carboxyl groups for bonding to amino groups.
Blood Components: Blood components may be either fixed or mobile. Fixed blood components are non-mobile blood components and include tissues, membrane receptors, interstitial proteins, fibrin proteins, collagens, platelets, endothelial cells, epithelial cells and their associated membrane and membraneous receptors, somatic body cells, skeletal and smooth muscle cells, neuronal components, osteocytes and osteoclasts and all body tissues especially those associated with the circulatory and lymphatic systems. Mobile blood components are blood components that do not have a fixed situs for any extended period of time, generally not exceeding 5, more usually one minute. These blood components are not membrane-associated and are present in the blood for extended periods of time and are present in a minimum concentration of at least 0.1 μg/ml. Mobile blood components include serum albumin, transferrin, ferritin and immunoglobulins such as IgM and IgG. The half-life of mobile blood components is at least about 12 hours.
Protective Groups: Protective groups are chemical moieties utilized to protect peptide derivatives from reacting with themselves. Various protective groups are disclosed herein and in U.S. Pat. No. 5,493,007, which is hereby incorporated by reference. Such protective groups include acetyl, fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (CBZ), and the like. The specific protected amino acids are depicted in Table 1.

TABLE 1

NATURAL AMINO ACIDS AND THEIR ABBREVIATIONS

	3-Letter	1-Letter
Name	Abbreviation	Abbreviation	Modified Amino Acids

Alanine	Ala	A	Fmoc-Ala-OH
Arginine	Arg	R	Fmoc-Arg(Pbf)-OH
Asparagine	Asn	N	Fmoc-Asn(Trt)-OH
Aspartic acid	Asp	D	Asp(tBu)-OH
Cysteine	Cys	C	Fmoc-Cys(Trt)
Glutamic acid	Glu	E	Fmoc-Glu(tBu)-OH
Glutamine	Gln	Q	Fmoc-Gln(Trt)-OH
Glycine	Gly	G	Fmoc-Gly-OH
Histidine	His	H	Fmoc-His(Trt)-OH
Isoleucine	Ile	I	Fmoc-Ile-OH
Leucine	Leu	L	Fmoc-Leu-OH
Lysine	Lys	Z	Boc-Lys(Aloc)-OH
Lysine	Lys	X	Fmoc-Lys(Aloc)-OH
Lysine	Lys	K	Fmoc-Lys(Mtt)-OH
Methionine	Met	M	Fmoc-Met-OH
Phenylalanine	Phe	F	Fmoc-Phe-OH
Proline	Pro	P	Fmoc-Pro-OH
Serine	Ser	S	Fmoc-Ser(tBu)-OH
Threonine	Thr	T	Fmoc-Thr(tBu)-OH
Tryptophan	Trp	W	Fmoc-Trp(Boc)-OH
Tyrosine	Tyr	Y	Boc-Tyr(tBu)-OH
Valine	Val	V	Fmoc-Val-OH

Linking Groups: Linking (spacer) groups are chemical moieties that link or connect reactive entities to antiviral or antifusogenic peptides. Linking groups may comprise one or more alkyl moeities, alkoxy moeity, alkenyl moeity, alkynyl moeity or amino moeity substituted by alkyl moeities, cycloalkyl moeity, polycyclic moeity, aryl moeity, polyaryl moeities, substituted aryl moeities, heterocyclic moeities, and substituted heterocyclic moeities. Linking groups may also comprise poly ethoxy amino acids, such as AEA ((2-amino) ethoxy acetic acid) or a preferred linking group AEEA ([2-(2-amino)ethoxy)]ethoxy acetic acid.
Sensitive Functional Groups—A sensitive functional group is a group of atoms that represents a potential reaction site on an antiviral and/or antifusogenic peptide. If present, a sensitive functional group may be chosen as the attachment point for the linker-reactive group modification. Sensitive functional groups include but are not limited to carboxyl, amino, thiol, and hydroxyl groups.
Modified Peptides—A modified peptide is an antiviral and/or antifusogenic peptide that has been modified by attaching a reactive group. The reactive group may be attached to the peptide either via a linking group, or optionally without using a linking group. It is also contemplated that one or more additional amino acids may be added to the peptide to facilitate the attachment of the reactive entity. Modified peptides may be administered in vivo such that conjugation with blood components occurs in vivo, or they may be first conjugated to blood components in vitro and the resulting conjugated peptide (as defined below) administered in vivo.
Conjugated Peptides—A conjugated peptide is a modified peptide that has been conjugated to a blood component via a covalent bond formed between the reactive group of the modified peptide and the functionalities of the blood component, with or without a linking group. As used throughout this application, the term “conjugated peptide” can be made more specific to refer to particular conjugated peptides, for example “conjugated DP178” or “conjugated DP107.”
Taking into account these definitions, the present invention takes advantage of the properties of existing anti-viral and antifusogenic peptides. The viruses that may be inhibited by the peptides include, but are not limited to all strains of viruses listed, e.g., in U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 at Tables V-VII and IX-XIV therein. These viruses include, e.g., human retroviruses, including HIV-1, HIV-2, and human T-lympocyte viruses (HTLV-I and HTLV-II), and non-human retroviruses, including bovine leukosis virus, feline sarcoma virus, feline leukemia virus, simian immunodeficiency virus (SIV), simian sarcoma virus, simian leukemia, and sheep progress pneumonia virus. Non-retroviral viruses may also be inhibited by the peptides of the present invention, including human respiratory syncytial virus (RSV), canine distemper virus, Newcastle Disease virus, human parainfluenza virus (HPIV), influenza viruses, measles viruses (MeV), Epstein-Barr viruses, hepatitis B viruses, and simian Mason-Pfizer viruses. Non-enveloped viruses may also be inhibited by the peptides of the present invention, and include, but are not limited to, picornaviruses such as polio viruses, hepatitis A virus, enteroviruses, echoviruses, coxsackie viruses, papovaviruses such as papilloma virus, parvoviruses, adenoviruses, and reoviruses.
As an example, the mechanism of action of HIV fusion peptides has been described as discussed in the background section of this application and antiviral and antifusogenic properties of the peptides have been well established. A synthetic peptide corresponding to the carboxyl-terminal ectodomain sequence (for instance, amino acid residues 643-678 of HIV-1 class B, of the LAI strain or residues 638-673 from similar strain as well as residues 558-595) has been shown to inhibit virus-mediated cell-cell fusion completely at low concentration. The fusion peptide competes with the leucine zipper region of the native viral gp41 thus resulting in the interference of the fusion/infection of the virus into the cell.
The focus of the present invention is to modify a selected anti-viral and/or antifusogenic peptide with the DAC (Drug Activity Complex) technology to confer to this peptide improved bio-availability, extended half-life and better distribution through selective conjugation of the peptide onto a protein carrier but without modifying the peptide's anti-viral properties. The carrier of choice (but not limited to) for this invention would be albumin conjugated through its free thiol by an anti-viral and/or antifusogenic peptide modified with a maleimide moiety.
Several peptide sequences have been described in the literature as highly potent for the prevention of HIV-1 fusion/infection. As examples, peptide DP 178 binds to a conformation of gp41 that is relevant for fusion. Thus in one embodiment of the invention, DP178 and DP178-like peptides are modified. Likewise, other embodiments of the invention include modification of DP107 and DP107-like peptide for use against HIV, as well as peptides analagous to DP107 and DP178 that are found in RSV, HPV, MeV and SIV viruses.

1. DP178 and DP107

A. DP178 Peptides
The DP178 peptide corresponds to amino acid residues 638 to 673 of the transmembrane protein gp41 from the HIV-1_LAIisolate, and has the 36 amino acid sequence (reading from amino to carboxy terminus):
(SEQ ID NO: 1)

NH₂-YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF-COOH
In addition to the full-length DP178 36-mer, the peptides of this invention include truncations of the DP178 peptide comprising peptides of between 3 and 36 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 36-mer polypeptide), These truncated peptides are shown in Tables 2 and 3.
In addition amino acid substitutions of the DP178 peptide are also within the scope of the invention. HIV-1 and HIV-2 enveloped proteins are structurally distinct, but there exists a striking amino acid conservation within the DP178-corresponding regions of HIV-1 and HIV-2. The amino acid conservation is of a periodic nature, suggesting some conservation of structure and/or function. Therefore, one possible class of amino acid substitutions would include those amino acid changes which are predicted to stabilize the structure of the DP178 peptides of the invention. Utilizing the DP178 and DP178 analog sequences described herein, the skilled artisan can readily compile DP178 consensus sequences and ascertain from these, conserved amino acid residues which would represent preferred amino acid substitutions.
The amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of the DP178 peptide sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to aspartic acid (D) amino acid substitution. Non-conserved substitutions consist of replacing one or more amino acids of the DP178 peptide sequence with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to valine (V) substitution.
Amino acid insertions of DP178 may consist of single amino acid residues or stretches of residues. The insertions may be made at the carboxy or amino terminal end of the DP178 or DP178 truncated peptides, as well as at a position internal to the peptide.
Such insertions will generally range from 2 to 15 amino acids in length. It is contemplated that insertions made at either the carboxy or amino terminus of the peptide of interest may be of a broader size range, with about 2 to about 50 amino acids being preferred. One or more such insertions may be introduced into DP178 or DP178 truncations, as long as such insertions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described above.
Preferred amino or carboxy terminal insertions are peptides ranging from about 2 to about 50 amino acid residues in length, corresponding to gp41 protein regions either amino to or carboxy to the actual DP178 gp41 amino acid sequence, respectively. Thus, a preferred amino terminal or carboxy terminal amino acid insertion would contain gp41 amino acid sequences found immediately amino to or carboxy to the DP178 region of the gp41 protein.
Deletions of DP178 or DP178 truncations are also within the scope of this invention. Such deletions consist of the removal of one or more amino acids from the DP178 or DP178-like peptide sequence, with the lower limit length of the resulting peptide sequence being 4 to 6 amino acids.
Such deletions may involve a single contiguous or greater than one discrete portion of the peptide sequences. One or more such deletions may be introduced into DP178 or DP178 truncations, as long as such deletions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described above.
B. DP107 Peptides
DP107 is a 38 amino acid peptide which exhibits potent antiviral activity, and corresponds to residues 558 to 595 of HIV-1_LAIisolate transmembrane (TM) gp41 glycoprotein, as shown here:
(SEQ ID NO: 2)

NH₂-NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ-COOH
In addition to the full-length DP107 38-mer, the DP107 peptides include truncations of the DP107 peptide comprising peptides of between 3 and 38 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 38-mer polypeptide), These peptides are shown in Tables 4 and 5, below.
In addition, amino acid substitutions of the DP178 peptide are also within the scope of the invention. As for DP178, there also exists a striking amino acid conservation within the DP107-corresponding regions of HIV-1 and HIV-2, again of a periodic nature, suggesting conservation of structure and/or function. Therefore, one possible class of amino acid substitutions includes those amino acid changes predicted to stabilize the structure of the DP107 peptides of the invention. Utilizing the DP107 and DP107 analog sequences described herein, the skilled artisan can readily compile DP107 consensus sequences and ascertain from these, conserved amino acid residues which would represent preferred amino acid substitutions.
The amino acid substitutions may be of a conserved or non-conserved nature. Conserved amino acid substitutions consist of replacing one or more amino acids of the DP107 peptide sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to aspartic acid (D) amino acid substitution. Non-conserved substitutions consist of replacing one or more amino acids of the DP107 peptide sequence with amino acids possessing dissimilar charge, size, and/or hydrophobicity characteristics, such as, for example, a glutamic acid (E) to valine (V) substitution.
Amino acid insertions may consist of single amino acid residues or stretches of residues. The insertions may be made at the carboxy or amino terminal end of the DP107 or DP107 truncated peptides, as well as at a position internal to the peptide.
Such insertions will generally range from 2 to 15 amino acids in length. It is contemplated that insertions made at either the carboxy or amino terminus of the peptide of interest may be of a broader size range, with about 2 to about 50 amino acids being preferred. One or more such insertions may be introduced into DP107 or DP107 truncations, as long as such insertions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs described above.
Preferred amino or carboxy terminal insertions are peptides ranging from about 2 to about 50 amino acid residues in length, corresponding to gp41 protein regions either amino to or carboxy to the actual DP107 gp41 amino acid sequence, respectively. Thus, a preferred amino terminal or carboxy terminal amino acid insertion would contain gp41 amino acid sequences found immediately amino to or carboxy to the DP107 region of the gp41 protein.
Deletions of DP107 or DP107 truncations are also within the scope of this invention. Such deletions consist of the removal of one or more amino acids from the DP107 or DP107-like peptide sequence, with the lower limit length of the resulting peptide sequence being 4 to 6 amino acids.
Such deletions may involve a single contiguous or greater than one discrete portion of the peptide sequences. One or more such deletions may be introduced into DP107 or DP107 truncations, as long as such deletions result in peptides which may still be recognized by the 107×178×4, ALLMOTI5 or PLZIP search motifs.
DP107 and DP107 truncations are more fully described in U.S. Pat. No. 5,656,480, which is incorporated herein by reference in its entirety

2. DP107 and DP178 Analogs

Peptides corresponding to analogs of the DP178, DP178 truncations, DP107 and DP107 truncation sequences of the invention, described, above, may be found in other viruses, including, for example, non-HIV-1 enveloped viruses, non-enveloped viruses and other non-viral organisms.
Such DP178 and DP107 analogs may, for example, correspond to peptide sequences present in transmembrane (“TM”) proteins of enveloped viruses and may, correspond to peptide sequences present in non enveloped and nonviral organisms. Such peptides may exhibit antifusogenic activity, antiviral activity, most particularly antiviral activity which is specific to the virus in which their native sequences are found, or may exhibit an ability to modulate intracellular processes involving coiled-coil peptide structures.
A. DP178 Analogs
DP178 analogs are peptides whose amino acid sequences are comprised of the amino acid sequences of peptide regions of, for example, other (i.e., other than HIV-1) viruses that correspond to the gp41 peptide region from which DP178 was derived. Such viruses may include, but are not limited to, other HIV-1 isolates and HIV-2 isolates.
DP178 analogs derived from the corresponding gp41 peptide region of other (i.e., non HIV-1LAI) HIV-1 isolates may include, for example, peptide sequences as shown below.

(SEQ ID NO: 3)

	NH2-YTNTIYTLLEESQNQQEKNEQELLELDKWASLWNWF-COOH

(SEQ ID NO: 4)

	NH2-YTGIIYNLLEESQNQQEKNEQELLELDKWANLWNWF-COOH

(SEQ ID NO: 5)

NH2-YTSLIYSLLEKSQIQQEKNEQELLELDKWASLWNWF-COOH

The peptides of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 are derived from HIV-1_SF2, HIV-1_RF, and HIV-1_MN, respectively. Other DP178 analogs include those derived from HIV-2, including the peptides of SEQ ID NO:6 and SEQ ID NO:7, which are derived from HIV-2_RODand HIV-2_NIHZ, respectively. Still other useful analogs include the peptides of SEQ ID NO:8 and SEQ ID NO:9, which have been demonstrated to exhibit anti-viral activity.
In the present invention, it is preferred that the DP178 analogs represent peptides whose amino acid sequences correspond to the DP178 region of the gp41 protein, it is also contemplated that the peptides disclosed herein may, additionally, include amino sequences, ranging from about 2 to about 50 amino acid residues in length, corresponding to gp41 protein regions either amino to or carboxy to the actual DP178 amino acid sequence.
Table 6 and Table 7 show some possible truncations of the HIV-2_NIHZDP178 analog, which may comprise peptides of between 3 and 36 amino acid residues (i.e., peptides ranging in size from a tripeptide to a 36-mer polypeptide). Peptide sequences in these tables are listed from amino (left) to carboxy (right) terminus.
B. Additional DP178 Analogs and DP107 Analogs
DP178 and DP107 analogs are recognized or identified, for example, by utilizing one or more of the 107×178×4, ALLMOTI5 or PLZIP computer-assisted search strategies described above. The search strategy identifies additional peptide regions which are predicted to have structural and/or amino acid sequence features similar to those of DP107 and/or DP178.
The search strategies are described fully in the example presented in Section 9 of U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459. While this search strategy is based, in part, on a primary amino acid motif deduced from DP107 and DP178, it is not based solely on searching for primary amino acid sequence homologies, as such protein sequence homologies exist within, but not between major groups of viruses. For example, primary amino acid sequence homology is high within the TM protein of different strains of HIV-1 or within the TM protein of different isolates of simian immunodeficiency virus (SIV).
The computer search strategy disclosed in U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 successfully identified regions of proteins similar to DP107 or DP178. This search strategy was designed to be used with a commercially-available sequence database package, preferably PC/Gene.
In U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459, a series of search motifs, the 107×178×4, ALLMOTI5 and PLZIP motifs, were designed and engineered to range in stringency from strict to broad, with 107×178×4 being preferred. The sequences identified via such search motifs, such as those listed in Tables V-XIV, of U.S. Pat. Nos. 6,013,263, 6,017,536 and 6,020,459 and included in this application by incorporation by reference, potentially exhibit antifusogenic, such as antiviral, activity, may additionally be useful in the identification of antifusogenic, such as antiviral, compounds.

3. Other Anti-Viral Peptides

A. Anti-RSV Peptides
Anti-RSV peptides include DP178 and/or DP107 analogs identified from corresponding peptide sequences in RSV which have further been identified to inhibit viral infection by RSV. Such peptides of interest include the peptides of Table 16 and peptides of SEQ ID NO:10 to SEQ ID NO:30. Of particular interest are the following peptides:

YTSVITIELSNIKENKCNGAKVKLIKQELDKYK	(SEQ ID NO: 14)

TSVITIELSNIKENKCNGAKVKLIKQELDKYKN	(SEQ ID NO: 15)

VITIELSNIKENKCNGAKVKLIKQELDKYKNAV	(SEQ ID NO: 16)

IALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK	(SEQ ID NO: 29)

The peptide of SEQ ID NO:10 is derived from the F2 region of RSV and was identified in U.S. Pat. Nos. 6,103,236 and 6,020,459 using the search motifs described as corresponding to DP107 and DP178 peptides (i.e., “DP107/178 like”). The peptides of SEQ ID NO:14 to SEQ ID NO:16 each have amino acid sequences contained within the peptide of SEQ ID NO:10 and each has been shown to exhibit anti-RSV activity, in particular, inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells at concentrations of less than 50 μg/ml.

The peptide of SEQ ID NO:11 is derived from the F1 region of RSV and was identified in U.S. Pat. Nos. 6,103,236 and 6,020,459 using the search motifs described as corresponding to DP107 (i.e., “DP107-like”). The peptide of SEQ ID NO:29 contains amino acid sequences contained within the peptide of SEQ ID NO:10 and likewise has been shown to exhibit anti-RSV activity, in particular, inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells at concentrations of less than 50 μg/ml.
B. Anti-HPIV Peptides
Anti-HPIV peptides include DP178 and/or DP107 analogs identified from corresponding peptide sequences in HPIV and which have further been identified to inhibit viral infection by HPIV. Such peptides of interest include the peptides of Table 17 and SEQ ID NO:31 to SEQ ID NO:62. Of particular interest are the following peptides:

(SEQ ID NO: 52)

	VEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLI

(SEQ ID NO: 58)

	RSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV

(SEQ ID NO: 35)

	NSVALDPIDISIELNKAKSDLEESKEWIRRSNQKL

(SEQ ID NO: 38)

	ALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI

(SEQ ID NO: 39)

	LDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIG

(SEQ ID NO: 40)

	DPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN

(SEQ ID NO: 41)

	PIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNW

(SEQ ID NO: 42)

IDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWH

The peptide of SEQ ID NO:31 is derived from the F1 region of HPIV-3 and was identified in U.S. Pat. Nos. 6,103,236 and 6,020,459 using the search motifs described as corresponding to DP107 (i.e., “DP107-like”). The peptides of SEQ ID NO:52 and SEQ ID NO:58 each have amino acid sequences contained within the peptide of SEQ ID NO:30 and each has been shown to exhibit anti-HPIV-3 activity, in particular, inhibiting fusion and syncytia formation between HPIV-3-infected Hep2 cells and uninfected CV-1W cells at concentrations of less than 1 μg/ml.
The peptide of SEQ ID NO:32 is also derived from the F1 region of HPIV-3 and was identified in U.S. Pat. Nos. 6,103,236 and 6,020,459 using the search motifs described as corresponding to DP178 (i.e., “DP178-like”). The peptides of SEQ ID NO:35 and SEQ ID NO:38 to SEQ ID NO:42 each have amino acid sequences contained within the peptide of SEQ ID NO:32 and each also has been shown to exhibit anti-HPIV-3 activity, in particular, inhibiting fusion and syncytia formation between HPIV-3-infected Hep2 cells and uninfected CV-1W cells at concentrations of less than 1 μg/ml.
C. Anti-MeV Peptides
Anti-MeV peptides are DP178 and/or DP107 analogs identified from corresponding peptide sequences in measles virus (MeV) which have further been identified to inhibit viral infection by the measles virus. Such peptides of particular interest include the peptides of Table 19 and peptides of SEQ ID NO:74 to SEQ ID NO:86. Of particular interest are the peptides listed below.

HRIDLGPPISLERLDVGTNLGNAIAKLEAKELLE	(SEQ ID NO: 77)

IDLGPPISLERLDVGTNLGNAIAKLEAKELLESS	(SEQ ID NO: 79)

LGPPISLERLDVGTNLGNAIAKLEAKELLESSDQ	(SEQ ID NO: 81)

PISLERLDVGTNLGNAIAKLEAKELLESSDQILR	(SEQ ID NO: 84)

Sequences derived from measles virus were identified in U.S. Pat. Nos. 6,103,236 and 6,020,459 using the search motifs described as corresponding to DP178 (i.e., “DP178-like”). The peptides of SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81 and SEQ ID NO:83 each have amino acid sequences so identified, and each has been shown to exhibit anti-MeV activity, in particular, inhibiting fusion and syncytia formation between MeV-infected Hep2 and uninfected Vero cells at concentrations of less than 1 μg/ml.

D. Anti-SIV Peptides
Anti-SIV peptides are DP178 and/or DP107 analogs identified from corresponding peptide sequences in SIV which have further been identified to inhibit viral infection by SIV. Such peptides of interest include the peptides of Table 18 and peptides of SEQ ID NO:63 to SEQ ID NO:73. Of particular interest are the following peptides:
(SEQ ID NO: 64)

WQEWERKVDFLEENITALLEEAQIQQEKNMYELQK

(SEQ ID NO: 65)

QEWERKVDFLEENITALLEEAQIQQEKNMYELQKL

(SEQ ID NO: 66)

EWERKVDFLEENITALLEEAQIQQEKNMYELQKLN

(SEQ ID NO: 67)

WERKVDFLEENITALLEEAQIQQEKNMYELQKLNS

(SEQ ID NO: 68)

ERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW

(SEQ ID NO: 69)

RKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD

(SEQ ID NO: 70)

KVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV

(SEQ ID NO: 71)

VDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF

(SEQ ID NO: 72)

DFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG

(SEQ ID NO: 73)

FLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN

Sequences derived from SIV transmembrane fusion protein were identified in U.S. Pat. Nos. 6,103,236 and 6,020,459 using the search motifs described as corresponding to DP178 (i.e., “DP178-like”). The peptides of SEQ ID NO:64 to SEQ ID NO:73 each have amino acid sequences so identified, and each has been shown to exhibit potent anti-SIV activity as crude peptides.

4. Modification of Anti-Viral and Antifusogenic Peptides

The invention contemplates modifying peptides that exhibit anti-viral and/or antifusogenic activity, including such modifications of DP-107 and DP-178 and analogs thereof. Such modified peptides can react with the available reactive functionalities on blood components via covalent linkages. The invention also relates to such modifications, such combinations with blood components, and methods for their use. These methods include extending the effective therapeutic life of the conjugated anti-viral peptides derivatives as compared to administration of the unconjugated peptides to a patient. The modified peptides are of a type designated as a DAC (Drug Affinity Complex) which comprises the anti-viral peptide molecule and a linking group together with a chemically reactive group capable of reaction with a reactive functionality of a mobile blood protein. By reaction with the blood component or protein the modified peptide, or DAC, may be delivered via the blood to appropriate sites or receptors.
To form covalent bonds with functionalities on the protein, one may use as a reactive group a wide variety of active carboxyl groups, particularly esters, where the hydroxyl moiety is physiologically acceptable at the levels required to modify the peptide. While a number of different hydroxyl groups may be employed in these reactive groups, the most convenient would be N-hydroxysuccinimide or (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS). In preferred embodiments of this invention, the functionality on the protein will be a thiol group and the reactive group will be a maleimido-containing group such as gamma-maleimide-butyralamide (GMBA) or maleimidopropionic acid (MPA)
Primary amines are the principal targets for NHS esters. Accessible α-amine groups present on the N-termini of proteins react with NHS esters. However, α-amino groups on a protein may not be desirable or available for the NHS coupling. While five amino acids have nitrogen in their side chains, only the ε-amine of lysine reacts significantly with NHS esters. An amide bond is formed when the NHS ester conjugation reaction reacts with primary amines releasing N-hydroxysuccinimide as demonstrated in the schematic below.
In the preferred embodiments of this invention, the functional group on this protein will be a thiol group and the chemically reactive group will be a maleimido-containing group such as MPA or GMBA (gamma-maleimide-butyralamide). The maleimido group is most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is kept between 6.5 and 7.4. At pH 7.0, the rate of reaction of maleimido groups with sulfhydryls is 1000-fold faster than with amines. A stable thioether linkage between the maleimido group and the sulfhydryl is formed which cannot be cleaved under physiological conditions, as demonstrated in the following schematic.
A. Specific Labeling.
Preferably, the modified peptides of this invention are designed to specifically react with thiol groups on mobile blood proteins. Such reaction is preferably established by covalent bonding of the peptide modified with a maleimide link (e.g. prepared from GMBS, MPA or other maleimides) to a thiol group on a mobile blood protein such as serum albumin or IgG.
Under certain circumstances, specific labeling with maleimides offers several advantages over non-specific labeling of mobile proteins with groups such as NHS and sulfo-NHS. Thiol groups are less abundant in vivo than amino groups. Therefore, the maleimide-modified peptides of this invention, i.e., maleimide peptides, will covalently bond to fewer proteins. For example, in albumin (the most abundant blood protein) there is only a single thiol group. Thus, peptide-maleimide-albumin conjugates will tend to comprise approximately a 1:1 molar ratio of peptide to albumin. In addition to albumin, IgG molecules (class II) also have free thiols. Since IgG molecules and serum albumin make up the majority of the soluble protein in blood they also make up the majority of the free thiol groups in blood that are available to covalently bond to maleimide-modified peptides.
Further, even among free thiol-containing blood proteins, including IgGs, specific labeling with maleimides leads to the preferential formation of peptide-maleimide-albumin conjugates, due to the unique characteristics of albumin itself. The single free thiol group of albumin, highly conserved among species, is located at amino acid residue 34 (Cys³⁴). It has been demonstrated recently that the Cys³⁴of albumin has increased reactivity relative to free thiols on other free thiol-containing proteins. This is due in part to the very low pK value of 5.5 for the Cys³⁴of albumin. This is much lower than typical pK values for cysteine residues in general, which are typically about 8. Due to this low pK, under normal physiological conditions Cys³⁴of albumin is predominantly in the ionized form, which dramatically increases its reactivity. In addition to the low pK value of Cys³⁴, another factor which enhances the reactivity of Cys³⁴is its location, which is in a crevice close to the surface of one loop of region V of albumin. This location makes Cys³⁴very available to ligands of all kinds, and is an important factor in Cys³⁴'s biological role as free radical trap and free thiol scavenger. These properties make Cys³⁴highly reactive with maleimide-peptides, and the reaction rate acceleration can be as much as 1000-fold relative to rates of reaction of maleimide-peptides with other free-thiol containing proteins.
Another advantage of peptide-maleimide-albumin conjugates is the reproducibility associated with the 1:1 loading of peptide to albumin specifically at Cys³⁴. Other techniques, such as glutaraldehyde, DCC, EDC and other chemical activations of, e.g, free amines, lack this selectivity. For example, albumin contains 52 lysine residues, 25-30 of which are located on the surface of albumin and therefore accessible for conjugation. Activating these lysine residues, or alternatively modifying peptides to couple through these lysine residues, results in a heterogenous population of conjugates. Even if 1:1 molar ratios of peptide to albumin are employed, the yield will consist of multiple conjugation products, some containing 0, 1, 2 or more peptides per albumin, and each having peptides randomly coupled at any one or more of the 25-30 available lysine sites. Given the numerous possible combinations, characterization of the exact composition and nature of each conjugate batch becomes difficult, and batch-to-batch reproducibility is all but impossible, making such conjugates less desirable as a therapeutic. Additionally, while it would seem that conjugation through lysine residues of albumin would at least have the advantage of delivering more therapeutic agent per albumin molecule, studies have shown that a 1:1 ratio of therapeutic agent to albumin is preferred. In an article by Stehle, et al., “The Loading Rate Determines Tumor Targeting properties of Methotrexate-Albumin Conjugates in Rats,” Anti-Cancer Drugs, Vol. 8, pp. 677-685 (1988), incorporated herein in its entirety, the authors report that a 1:1 ratio of the anti-cancer methotrexate to albumin conjugated via glutaraldehyde gave the most promising results. These conjugates were preferentially taken up by tumor cells, whereas conjugates bearing 5:1 to 20:1 methotrexate molecules had altered HPLC profiles and were quickly taken up by the liver in vivo. It is postulated that at these higher ratios, conformational changes to albumin diminish its effectiveness as a therapeutic carrier.
Through controlled administration of maleimide-peptides in vivo, one can control the specific labeling of albumin and IgG in vivo. In typical administrations, 80-90% of the administered maleimide-peptides will label albumin and less than 5% will label IgG. Trace labeling of free thiols such as glutathione will also occur. Such specific labeling is preferred for in vivo use as it permits an accurate calculation of the estimated half-life of the administered agent.
In addition to providing controlled specific in vivo labeling, maleimide-peptides can provide specific labeling of serum albumin and IgG ex vivo. Such ex vivo labeling involves the addition of maleimide-peptides to blood, serum or saline solution containing serum albumin and/or IgG. Once conjugation has occurred ex vivo with the maleimide-peptides, the blood, serum or saline solution can be readministered to the patient's blood for in vivo treatment.
In contrast to NHS-peptides, maleimide-peptides are generally quite stable in the presence of aqueous solutions and in the presence of free amines. Since maleimide-peptides will only react with free thiols, protective groups are generally not necessary to prevent the maleimide-peptides from reacting with itself. In addition, the increased stability of the modified peptide permits the use of further purification steps such as HPLC to prepare highly purified products suitable for in vivo use. Lastly, the increased chemical stability provides a product with a longer shelf life.
B. Non-Specific Labeling.
The anti-viral peptides of the invention may also be modified for non-specific labeling of blood components. Bonds to amino groups will also be employed, particularly with the formation of amide bonds for non-specific labeling. To form such bonds, one may use as a chemically reactive group a wide variety of active carboxyl groups, particularly esters, where the hydroxyl moiety is physiologically acceptable at the levels required. While a number of different hydroxyl groups may be employed in these linking agents, the most convenient would be N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS).
Other linking agents which may be utilized are described in U.S. Pat. No. 5,612,034, which is hereby incorporated herein.
The various sites with which the chemically reactive group of the modified peptides may react in vivo include cells, particularly red blood cells (erythrocytes) and platelets, and proteins, such as immunoglobulins, including IgG and IgM, serum albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding protein, α-2-macroglobulin, and the like. Those receptors with which the modified peptides react, which are not long-lived, will generally be eliminated from the human host within about three days. The proteins indicated above (including the proteins of the cells) will remain at least three days, and may remain five days or more (usually not exceeding 60 days, more usually not exceeding 30 days) particularly as to the half life, based on the concentration in the blood.
For the most part, reaction will be with mobile components in the blood, particularly blood proteins and cells, more particularly blood proteins and erythrocytes. By “mobile” is intended that the component does not have a fixed situs for any extended period of time, generally not exceeding 5 minutes, more usually one minute, although some of the blood component may be relatively stationary for extended periods of time. Initially, there will be a relatively heterogeneous population of functionalized proteins and cells. However, for the most part, the population within a few days will vary substantially from the initial population, depending upon the half-life of the functionalized proteins in the blood stream. Therefore, usually within about three days or more, IgG will become the predominant functionalized protein in the blood stream.
Usually, by day 5 post-administration, IgG, serum albumin and erythrocytes will be at least about 60 mole %, usually at least about 75 mole %, of the conjugated components in blood, with IgG, IgM (to a substantially lesser extent) and serum albumin being at least about 50 mole %, usually at least about 75 mole %, more usually at least about 80 mole %, of the non-cellular conjugated components.
The desired conjugates of non-specific modified peptides to blood components may be prepared in vivo by administration of the modified peptides to the patient, which may be a human or other mammal. The administration may be done in the form of a bolus or introduced slowly over time by infusion using metered flow or the like.
If desired, the subject conjugates may also be prepared ex vivo by combining blood with modified peptides of the present invention, allowing covalent bonding of the modified peptides to reactive functionalities on blood components and then returning or administering the conjugated blood to the host. Moreover, the above may also be accomplished by first purifying an individual blood component or limited number of components, such as red blood cells, immunoglobulins, serum albumin, or the like, and combining the component or components ex vivo with the chemically reactive modified peptides. The functionalized blood or blood component may then be returned to the host to provide in vivo the subject therapeutically effective conjugates. The blood also may be treated to prevent coagulation during handling ex vivo.

5. Synthesis of Modified Anti-Viral and Anti-Fusogenic Peptides

A. Peptide Synthesis
Anti-viral and/or anti-fusogenic peptides according to the present invention may be synthesized by standard methods of solid phase peptide chemistry known to those of ordinary skill in the art. For example, peptides may be synthesized by solid phase chemistry techniques following the procedures described by Steward and Young (Steward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984) using an Applied Biosystem synthesizer. Similarly, multiple peptide fragments may be synthesized then linked together to form larger peptides. These synthetic peptides can also be made with amino acid substitutions at specific locations.
For solid phase peptide synthesis, a summary of the many techniques may be found in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis see G. Schroder and K. Lupke, The Peptides, Vol. 1, Acacemic Press (New York). In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid is then either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected and under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth.
After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently to afford the final polypeptide. By simple modification of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide.
A particularly preferred method of preparing compounds of the present invention involves solid phase peptide synthesis wherein the amino acid α-N-terminal is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, and the like. The 9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the peptides of the present invention. Other preferred side chain protecting groups are, for side chain amino groups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for asparticacid and glutamic acid, benzyl and t-butyl and for cysteine, triphenylmethyl (trityl).
In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used. The preferred solid support for synthesis of α-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene). The preferred solid support for α-C-terminal amide peptides is the 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin available from Applied Biosystems (Foster City, Calif.). The α-C-terminal amino acid is coupled to the resin by means of N,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC) or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (HBTU), with or without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediated coupling for from about 1 to about 24 hours at a temperature of between 10° and 50° C. in a solvent such as dichloromethane or DMF.
When the solid support is 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine, preferably piperidine, prior to coupling with the α-C-terminal amino acid as described above. The preferred method for coupling to the deprotected 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate (HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art. In a preferred embodiment, the α-N-terminal amino acids of the growing peptide chain are protected with Fmoc. The removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in about 3-fold molar excess, and the coupling is preferably carried out in DMF. The coupling agent is normally O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.).
At the end of the solid phase synthesis, the polypeptide is removed from the resin and deprotected, either in successively or in a single operation. Removal of the polypeptide and deprotection can be accomplished in a single operation by treating the resin-bound polypeptide with a cleavage reagent comprising thioanisole, water, ethanedithiol and trifluoroacetic acid. In cases wherein the α-C-terminal of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide may be removed by transesterification, e.g. with methanol, followed by aminolysis or by direct transamidation. The protected peptide may be purified at this point or taken to the next step directly. The removal of the side chain protecting groups is accomplished using the cleavage cocktail described above. The fully deprotected peptide is purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivitized polystyrene-divinylbenzene (for example, Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography, e.g. on Sephadex G-25, LH-20 or countercurrent distribution; high performance liquid chromatography (HPLC), especially reverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.
Molecular weights of these ITPs are determined using Fast Atom Bombardment (FAB) Mass Spectroscopy.

(1) N-Terminal Protective Groups

As discussed above, the term “N-protecting group” refers to those groups intended to protect the α-N-terminal of an amino acid or peptide or to otherwise protect the amino group of an amino acid or peptide against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups In Organic Synthesis,” (John Wiley & Sons, New York (1981)), which is hereby incorporated by reference. Additionally, protecting groups can be used as pro-drugs which are readily cleaved in vivo, for example, by enzymatic hydrolysis, to release the biologically active parent. α-N-protecting groups comprise loweralkanoyl groups such as formyl, acetyl (“Ac”), propionyl, pivaloyl, t-butylacetyl and the like; other acyl groups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups such as trimethylsilyl and the like.

(2) Carboxy Protective Groups

As discussed above, the term “carboxy protecting group” refers to a carboxylic acid protecting ester or amide group employed to block or protect the carboxylic acid functionality while the reactions involving other functional sites of the compound are performed. Carboxy protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis” pp. 152-186 (1981), which is hereby incorporated by reference. Additionally, a carboxy protecting group can be used as a pro-drug whereby the carboxy protecting group can be readily cleaved in vivo, for example by enzymatic hydrolysis, to release the biologically active parent. Such carboxy protecting groups are well known to those skilled in the art, having been extensively used in the protection of carboxyl groups in the penicillin and cephalosporin fields as described in U.S. Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which are hereby incorporated herein by reference. Representative carboxy protecting groups are C₁-C₈loweralkyl (e.g., methyl, ethyl or t-butyl and the like); arylalkyl such as phenethyl or benzyl and substituted derivatives thereof such as alkoxybenzyl or nitrobenzyl groups and the like; arylalkenyl such as phenylethenyl and the like; aryl and substituted derivatives thereofsuch as 5-indanyl and the like; dialkylaminoalkyl such as dimethylaminoethyl and the like); alkanoyloxyalkyl groups such as acetoxymethyl, butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl, 1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl, pivaloyloxymethyl, propionyloxymethyl and the like; cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl and the like; arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl, 2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl or cycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl, cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like; alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such as methoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl, 1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and the like; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl, 2-(5-indanyloxycarbonyloxy)ethyl and the like; alkoxyalkylcarbonyloxyalkyl such as 2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like; arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl and the like; arylalkenyloxycarbonyloxyalkyl such as 2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like; alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and the like; alkylaminocarbonylaminoalkyl such as methylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such as acetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as 4-methylpiperazinylcarbonyloxymethyl and the like; dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl, diethylaminocarbonylmethyl and the like; (5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like; and (5-phenyl-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.
Representative amide carboxy protecting groups are aminocarbonyl and loweralkylaminocarbonyl groups.
Preferred carboxy-protected compounds of the invention are compounds wherein the protected carboxy group is a loweralkyl, cycloalkyl or arylalkyl ester, for example, methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester and the like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester. Preferred amide carboxy protecting groups are loweralkylaminocarbonyl groups. For example, aspartic acid may be protected at the α-C-terminal by an acid labile group (e.g. t-butyl) and protected at the β-C-terminal by a hydrogenation labile group (e.g. benzyl) then deprotected selectively during synthesis.
B. Peptide Modification
The manner of producing the modified peptides of the present invention will vary widely, depending upon the nature of the various elements comprising the peptide. The synthetic procedures will be selected so as to be simple, provide for high yields, and allow for a highly purified stable product. Normally, the chemically reactive group will be created at the last stage of the synthesis, for example, with a carboxyl group, esterification to form an active ester. Specific methods for the production of modified peptides of the present invention are described below.
Specifically, the selected peptide is first assayed for anti-viral activity, and then is modified with the linking group only at either the N-terminus, C-terminus or interior of the peptide. The anti-viral activity of this modified peptide-linking group is then assayed. If the anti-viral activity is not reduced dramatically (i.e., reduced less than 10-fold), then the stability of the modified peptide-linking group is measured by its in vivo lifetime. If the stability is not improved to a desired level, then the peptide is modified at an alternative site, and the procedure is repeated until a desired level of anti-viral and stability is achieved.
More specifically, each peptide selected to undergo modification with a linker and a reactive entity group will be modified according to the following criteria: if a terminal carboxylic group is available on the peptide and is not critical for the retention of anti-viral activity, and no other sensitive functional group is present on the peptide, then the carboxylic acid will be chosen as attachment point for the linker-reactive group modification. If the terminal carboxylic group is involved in anti-viral activity, or if no carboxylic acids are available, then any other sensitive functional group not critical for the retention of anti-viral activity will be selected as the attachment point for the linker-reactive entity modification. If several sensitive functional groups are available on a a peptide, a combination of protecting groups will be used in such a way that after addition of the linker/reactive entity and deprotection of all the protected sensitive functional groups, retention of anti-viral activity is still obtained. If no sensitive functional groups are available on the peptide, or if a simpler modification route is desired, synthetic efforts will allow for a modification of the original peptide in such a way that retention of anti-viral is maintained. In this case the modification will occur at the opposite end of the peptide
An NHS derivative may be synthesized from a carboxylic acid in absence of other sensitive functional groups in the peptide. Specifically, such a peptide is reacted with N-hydroxysuccinimide in anhydrous CH₂Cl₂and EDC, and the product is purified by chromatography or recrystallized from the appropriate solvent system to give the NHS derivative.
Alternatively, an NHS derivative may be synthesized from a peptide that contains an amino and/or thiol group and a carboxylic acid. When a free amino or thiol group is present in the molecule, it is preferable to protect these sensitive functional groups prior to perform the addition of the NHS derivative. For instance, if the molecule contains a free amino group, a transformation of the amine into a Fmoc or preferably into a tBoc protected amine is necessary prior to perform the chemistry described above. The amine functionality will not be deprotected after preparation of the NHS derivative. Therefore this method applies only to a compound whose amine group is not required to be freed to induce the desired anti-viral effect. If the amino group needs to be freed to retain the original properties of the molecule, then another type of chemistry described below has to be performed.
In addition, an NHS derivative may be synthesized from a peptide containing an amino or a thiol group and no carboxylic acid. When the selected molecule contains no carboxylic acid, an array of bifunctional linkers can be used to convert the molecule into a reactive NHS derivative. For instance, ethylene glycol-bis(succinimydylsuccinate) (EGS) and triethylamine dissolved in DMF and added to the free amino containing molecule (with a ratio of 10:1 in favor of EGS) will produce the mono NHS derivative. To produce an NHS derivative from a thiol derivatized molecule, one can use N-[-maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine in DMF. The maleimido group will react with the free thiol and the NHS derivative will be purified from the reaction mixture by chromatography on silica or by HPLC.
An NHS derivative may also be synthesized from a peptide containing multiple sensitive functional groups. Each case will have to be analyzed and solved in a different manner. However, thanks to the large array of protecting groups and bifunctional linkers that are commercially available, this invention is applicable to any peptide with preferably one chemical step only to modify the peptide (as described above) or two steps (as described above involving prior protection of a sensitive group) or three steps (protection, activation and deprotection). Under exceptional circumstances only, would multiple steps (beyond three steps) synthesis be required to transform a peptide into an active NHS or maleimide derivative.
A maleimide derivative may also be synthesized from a peptide containing a free amino group and a free carboxylic acid. To produce a maleimide derivative from a amino derivatized molecule, one can use N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine in DMF. The succinimide ester group will react with the free amino and the maleimide derivative will be purified from the reaction mixture by crystallization or by chromatography on silica or by HPLC.
Finally, a maleimide derivative may be synthesized from a peptide containing multiple other sensitive functional groups and no free carboxylic acids. When the selected molecule contains no carboxylic acid, an array of bifunctional crosslinking reagents can be used to convert the molecule into a reactive NHS derivative. For instance maleimidopropionic acid (MPA) can be coupled to the free amine to produce a maleimide derivative through reaction of the free amine with the carboxylic group of MPA using HBTU/HOBt/DIEA activation in DMF.
Many other commercially available heterobifunctional crosslinking reagents can alternatively be used when needed. A large number of bifunctional compounds are available for linking to entities. Illustrative reagents include: azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyldithio)propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxy sulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3′-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

6. Uses of Modified Anti-Viral Peptides

Modified anti-viral peptides of the invention may be used as a therapeutic agent in the treatment of patients who are suffering from viral infection, and can be administered to patients according to the methods described below and other methods known in the art. Effective therapeutic dosages of the modified peptides may be determined through procedures well known by those in the art and will take into consideration any concerns over potential toxicity of the peptide.
The modified peptides can also be administered prophylactically to previously uninfected individuals. This can be advantageous in cases where an individual has been subjected to a high risk of exposure to a virus, as can occur when individual has been in contact with an infected individual where there is a high risk of viral transmission. This can be expecially advantageous where there is known cure for the virus, such as the HIV virus. As a example, prophylactic administration of a modified anti-HIV peptide would be advantageous in a situation where a health care worker has been exposed to blood from an HIV-infected individual, or in other situations where an individual engaged in high-risk activities that potentially expose that individual to the HIV virus.

7. Administration of Modified Anti-Viral and Anti-Fusogenic Peptides

Generally, the modified peptides will be administered in a physiologically acceptable medium, e.g. deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetable oil, or the like. Other additives which may be included include buffers, where the media are generally buffered at a pH in the range of about 5 to 10, where the buffer will generally range in concentration from about 50 to 250 mM, salt, where the concentration of salt will generally range from about 5 to 500 mM, physiologically acceptable stabilizers, and the like. The compositions may be lyophilized for convenient storage and transport.
The subject modified peptides will for the most part be administered parenterally, such as intravenously (IV), intraarterially (IA), intramuscularly (IM), subcutaneously (SC), or the like. Administration may in appropriate situations be by transfusion. In some instances, where reaction of the functional group is relatively slow, administration may be oral, nasal, rectal, transdermal or aerosol, where the nature of the conjugate allows for transfer to the vascular system. Usually a single injection will be employed although more than one injection may be used, if desired. The modified peptides may be administered by any convenient means, including syringe, trocar, catheter, or the like.
The particular manner of administration will vary depending upon the amount to be administered, whether a single bolus or continuous administration, or the like. Preferably, the administration will be intravascularly, where the site of introduction is not critical to this invention, preferably at a site where there is rapid blood flow, e.g., intravenously, peripheral or central vein. Other routes may find use where the administration is coupled with slow release techniques or a protective matrix. The intent is that the modified peptide be effectively distributed in the blood, so as to be able to react with the blood components. The concentration of the conjugate will vary widely, generally ranging from about 1 pg/ml to 50 mg/ml. The total administered intravascularly will generally be in the range of about 0.1 mg/ml to about 10 mg/ml, more usually about 1 mg/ml to about 5 mg/ml.
By bonding to long-lived components of the blood, such as immunoglobulin, serum albumin, red blood cells and platelets, a number of advantages ensue. The activity of the peptide is extended for days to weeks. Only one administration need be given during this period of time. Greater specificity can be achieved, since the active compound will be primarily bound to large molecules, where it is less likely to be taken up intracellularly to interfere with other physiological processes.

8. Monitoring the Presence of Modified Peptides

The blood of the mammalian host may be monitored for the presence of the modified peptide compound one or more times. By taking a portion or sample of the blood of the host, one may determine whether the peptide has become bound to the long-lived blood components in sufficient amount to be therapeutically active and, thereafter, the level of the peptide compound in the blood. If desired, one may also determine to which of the blood components the peptide is bound. This is particularly important when using non-specific modified peptides. For specific maleimide-modified peptides, it is much simpler to calculate the half life of serum albumin and IgG.
A. Immuno Assays
Another aspect of this invention relates to methods for determining the concentration of the anti-viral peptides and/or analogs, or their derivatives and conjugates in biological samples (such as blood) using antibodies specific for the peptides, peptide analogs or their derivatives and conjugates, and to the use of such antibodies as a treatment for toxicity potentially associated with such peptides, analogs, and/or their derivatives or conjugates. This is advantageous because the increased stability and life of the peptides in vivo in the patient might lead to novel problems during treatment, including increased possibility for toxicity.
The use of anti-therapeutic agent antibodies, either monoclonal or polyclonal, having specificity for a particular peptide, peptide analog or derivative thereof, can assist in mediating any such problem. The antibody may be generated or derived from a host immunized with the particular peptide, analog or derivative thereof, or with an immunogenic fragment of the agent, or a synthesized immunogen corresponding to an antigenic determinant of the agent. Preferred antibodies will have high specificity and affinity for native, modified and conjugated forms of the peptide, peptide analog or derivative. Such antibodies can also be labeled with enzymes, fluorochromes, or radiolables.
Antibodies specific for modified peptides may be produced by using purified peptides for the induction of peptide-specific antibodies. By induction of antibodies, it is intended not only the stimulation of an immune response by injection into animals, but analogous steps in the production of synthetic antibodies or other specific binding molecules such as screening of recombinant immunoglobulin libraries. Both monoclonal and polyclonal antibodies can be produced by procedures well known in the art.
The anti-peptide antibodies may be used to treat toxicity induced by administration of the modified peptide, analog or derivative thereof, and may be used ex vivo or in vivo. Ex vivo methods would include immuno-dialysis treatment for toxicity employing anti-therapeutic agent antibodies fixed to solid supports. In vivo methods include administration of anti-therapeutic agent antibodies in amounts effective to induce clearance of antibody-agent complexes.
The antibodies may be used to remove the modified peptides, analogs or derivatives thereof, and conjugates thereof, from a patient's blood ex vivo by contacting the blood with the antibodies under sterile conditions. For example, the antibodies can be fixed or otherwise immobilized on a column matrix and the patient's blood can be removed from the patient and passed over the matrix. The modified peptide, peptide analogs, derivatives or conjugates will bind to the antibodies and the blood containing a low concentration of peptide, analog, derivative or conjugate, then may be returned to the patient's circulatory system. The amount of peptide compound removed can be controlled by adjusting the pressure and flow rate.
Preferential removal of the peptides, analogs, derivatives and conjugates from the plasma component of a patient's blood can be effected, for example, by the use of a semipermeable membrane, or by otherwise first separating the plasma component from the cellular component by ways known in the art prior to passing the plasma component over a matrix containing the anti-therapeutic antibodies. Alternatively the preferential removal of peptide-conjugated blood cells, including red blood cells, can be effected by collecting and concentrating the blood cells in the patient's blood and contacting those cells with fixed anti-therapeutic antibodies to the exclusion of the serum component of the patient's blood.
The anti-therapeutic antibodies can be administered in vivo, parenterally, to a patient that has received the peptide, analogs, derivatives or conjugates for treatment. The antibodies will bind peptide compounds and conjugates. Once bound the peptide activity will be hindered if not completely blocked thereby reducing the biologically effective concentration of peptide compound in the patient's bloodstream and minimizing harmful side effects. In addition, the bound antibody—peptide complex will facilitate clearance of the peptide compounds and conjugates from the patient's blood stream.
The invention having been fully described can be further appreciated and understood with reference to the following non-limiting examples.

EXAMPLE 1

Preparation of a Modified DP178—Synthesis of YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFK(MPA)-NH₂

In this example, DP178 (SEQ ID NO:1) is synthesized and modified to include a linker and maleimide group according to the following synthesis scheme. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, DP178 is a potent inhibitor of HIV-1, and inhibits both cell-induced syncytia formation between HIV-1 infected and uninfected cells and infection of uninfected cells be cell-free HIV-1 virus.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH; Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). At the end of the synthesis. The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 run to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 2

Preparation of a Modified DP107—Synthesis of

NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQK(MPA)NH₂
In this example, DP107 (SEQ ID NO:2) is synthesized and modified to include a linker and maleimide group according to the following synthesis scheme. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, DP107 exhibits potent antiviral activity against HIV.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-His(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH,They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). At the end of the synthesis. The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 3

Preparation of a Modified anti-RSV Peptide (C Terminal)
In this example, the peptide VITIELSNIKENKCNGAKVKLIKQELDKYKNAV (SEQ ID NO:16) is modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, the native sequence (SEQ ID NO.) inhibits viral infection of respiratory syncytial virus (RSV), including inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 4

Preparation of a Modified Anti-RSV Peptide (T-N Terminal)

In this example, the peptide VITIELSNIKENKCNGAKVKLIKQELDKYKNAV (SEQ ID NO:17), which corresponds to the peptide of SEQ ID NO:16 but where a Cysteine (C) has been substituted for the Methionine (M), residue is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, the native sequence (SEQ ID NO:16) inhibits viral infection of respiratory syncytial virus (RSV), including inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et2O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 5

Preparation of a Modified Anti-RSV Peptide

In this example, the peptide SEQ ID NO:14 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:14 inhibits viral infection of respiratory syncytial virus (RSV), including inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 6 (T-143)

Preparation of a Modified Anti-RSV Peptide

In this example, the peptide SEQ ID NO:15 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:15 inhibits viral infection of respiratory syncytial virus (RSV), including inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells.
Solid phase peptide synthesis of the modified peptide analog on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 7

Preparation of a Modified Anti-RSV Peptide (C Terminal)

In this example, the peptide SEQ ID NO:17), which corresponds to SEQ ID NO:16 with a cysteine (C) substituted for the Methionine (M), is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, the native sequence SEQ ID NO:16. inhibits viral infection of respiratory syncytial virus (RSV), including inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 8

Preparation of a Modified Anti-RSV Peptide

In this example, the peptide SEQ ID NO:29. is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:29 inhibits viral infection of respiratory syncytial virus (RSV), including inhibiting fusion and syncytia formation between RSV-infected and uninfected Hep-2 cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ile-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3. times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian 30 Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide

EXAMPLE 9 (T-173)

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:52. is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:52 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 10

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:58 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:58 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (1 8:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 11

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:35 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:35 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 12

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:38 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO: 38 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide analog on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH BOC-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (VN) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 13

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:39 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:39 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1 W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 14

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:40 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO. inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Asp(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 15

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:41 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:41 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH Boc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 16

Preparation of a Modified Anti-HPIV Peptide

In this example, the peptide SEQ ID NO:42 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:42 inhibits viral infection of human parainfluenza virus 3 (HPIV3), including inhibiting fusion and syncytia formation between HPIV3-infected Hep2 cells and uninfected CV-1W cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-His(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC

EXAMPLE 17

Preparation of a Modified Anti-MeV Peptide

In this example, the peptide SEQ ID NO:77. is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:77 inhibits viral infection of measles virus (MeV), including inhibiting fusion and syncytia formation between MeV-infected and uninfected Vero cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Boc)OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide. (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 18

Preparation of a Modified Anti-MeV Peptide

In this example, the peptide SEQ ID NO:79 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:79 inhibits viral infection of measles virus (MeV), including inhibiting fusion and syncytia formation between MeV-infected and uninfected Vero cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ile-OH, They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC

EXAMPLE 19

Preparation of a Modified Anti-MeV Peptide

In this example, the peptide SEQ ID NO:81 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO: 79 inhibits viral infection of measles virus (MeV), including inhibiting fusion and syncytia formation between MeV-infected and uninfected Vero cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Leu-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (VN) piperidine in N,N-dimethylformrnamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 20

Preparation of a Modified Anti-MeV Peptide

In this example, the peptide SEQ ID NO:84 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:84 inhibits viral infection of measles virus (MeV), including inhibiting fusion and syncytia formation between MeV-infected and uninfected Vero cells.
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Pro-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 21

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:64 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:64. exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 22

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:65 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:65 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (VN) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 23

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:66 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:66 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Glu(tBu)-OH Boc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 24

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:67 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:67 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Trp(Boc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylfQrmarnide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 25

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:68 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:68 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(tBu)-OH Boc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 26

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:69 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:69 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 27

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:70. is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:70 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Boc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 28

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:71 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:71 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH, Fmoc-Val-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 29

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:72 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:72 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asp(tBu)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.

EXAMPLE 30

Preparation of a Modified Anti-SIV Peptide

In this example, the peptide SEQ ID NO:73 is synthesized and modified to include a linker and maleimide group according to the synthesis scheme set forth below. As reported in U.S. Pat. Nos. 6,013,236 and 6,020,459, SEQ ID NO:73 exhibits potent antiviral activity as a crude peptide against simian immunodeficiency virus (SIV).
Solid phase peptide synthesis of the modified peptide on a 100 μmole scale is performed using manual solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved using a solution of 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl₃(6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Between every coupling, the resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II) at λ214 and 254 nm to afford the desired modified peptide (i.e., DAC) in >95% purity, as determined by RP-HPLC.
While certain embodiments of the invention have been described and exemplified, those having ordinary skill in the art will understand that the invention is not intended to be limited to the specifics of any of these embodiments, but is rather defined by the accompanying claims.

TABLE 2

DP178 CARBOXY TRUNCATIONS

	YTS

	YTSL

	YTSLI

	YTSLIH

	YTSLIHS

	YTSLIHSL

	YTSLIHSLI

	YTSLIHSLIE

	YTSLIHSLIEE

	YTSLIHSLIEES

	YTSLIHSLIEESQ

	YTSLIHSLIEESQN

	YTSLIHSLIEESQNQ

	YTSLIHSLIEESQNQQ

	YTSLIHSLIEESQNQQE

	YTSLIHSLIEESQNQQEK

	YTSLIHSLIEESQNQQEKN

	YTSLIHSLIEESQNQQEKNE

	YTSLIHSLIEESQNQQEKNEQ

	YTSLIHSLIEESQNQQEKNEQE

	YTSLIHSLIEESQNQQEKNEQEL

	YTSLIHSLIEESQNQQEKNEQELL

	YTSLIHSLIEESQNQQEKNEQELLE

	YTSLIHSLIEESQNQQEKNEQELLEL

	YTSLIHSLIEESQNQQEKNEQELLELD

	YTSLIHSLIEESQNQQEKNEQELLELDK

	YTSLIHSLIEESQNQQEKNEQELLELDKW

	YTSLIHSLIEESQNQQEKNEQELLELDKWA

	YTSLIHSLIEESQNQQEKNEQELLELDKWAS

	YTSLIHSLIEESQNQQEKNEQELLELDKWASL

	YTSLIHSLIEESQNQQEKNEQELLELDKWASLW

	YTSLIHSLIEESQNQQEKNEQELLELDKWASLWN

	YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNW

	YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

The one letter amino acid code of Table 1 is used.

TABLE 3

DP178 AMINO TRUNCATIONS

	NWF

	WNWF

	LWNWF

	SLWNWF

	ASLWNWF

	WASLWNWF

	KWASLWNWF

	DKWASLWNWF

	LDKWASLWNWF

	ELDKWASLWNWF

	LELDKWASLWNWF

	LLELDKWASLWNWF

	ELLELDKWASLWNWF

	QELLELDKWASLWNWF

	EQELLELDKWASLWNWF

	NEQELLELDKWASLWNWF

	KNEQELLELDKWASLWNWF

	EKNEQELLELDKWASLWNWF

	QEKNEQELLELDKWASLWNWF

	QQEKNEQELLELDKWASLWNWF

	NQQEKNEQELLELDKWASLWNWF

	QNQQEKNEQELLELDKWASLWNWF

	SQNQQEKNEQELLELDKWASLWNWF

	ESQNQQEKNEQELLELDKWASLWNWF

	EESQNQQEKNEQELLELDKWASLWNWF

	IEESQNQQEKNEQELLELDKWASLWNWF

	LIEESQNQQEKNEQELLELDKWASLWNWF

	SLIEESQNQQEKNEQELLELDKWASLWNWF

	HSLIEESQNQQEKNEQELLELDKWASLWNWF

	IHSLIEESQNQQEKNEQELLELDKWASLWNWF

	LIHSLIEESQNQQEKNEQELLELDKWASLWNWF

	SLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

	TSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

	YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

The one letter amino acid code of Table 1 is used.

TABLE 4

DP107 CARBOXY TRUNCATIONS

	NNL

	NNLL

	NNLLR

	NNLLRA

	NNLLRAI

	NNLLRAIE

	NNLLRAIEA

	NNLLRAIEAQ

	NNLLRAIEAQQ

	NNLLRAIEAQQH

	NNLLRAIEAQQHL

	NNLLRAIEAQQHLL

	NNLLRAIEAQQHLLQ

	NNLLRAIEAQQHLLQL

	NNLLRAIEAQQHLLQLT

	NNLLRAIEAQQHLLQLTV

	NNLLRAIEAQQHLLQLTVW

	NNLLRAIEAQQHLLQLTVWQ

	NNLLRAIEAQQHLLQLTVWQI

	NNLLRAIEAQQHLLQLTVWQIK

	NNLLRAIEAQQHLLQLTVWQIKQ

	NNLLRAIEAQQHLLQLTVWQIKQL

	NNLLRAIEAQQHLLQLTVWQIKQLQ

	NNLLRAIEAQQHLLQLTVWQIKQLQA

	NNLLRAIEAQQHLLQLTVWQIKQLQAR

	NNLLRAIEAQQHLLQLTVWQIKQLQARI

	NNLLRAIEAQQHLLQLTVWQIKQLQARIL

	NNLLRAIEAQQHLLQLTVWQIKQLQARILA

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAV

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVE

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVER

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERY

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYL

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLK

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKD

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

The one letter amino acid code of Table 1 is used.

TABLE 5

DP107 AMINO TRUNCATIONS

	KDQ

	LKDQ

	YLKDQ

	RYLKDQ

	ERYLKDQ

	VERYLKDQ

	AVERYLKDQ

	LAVERYLKDQ

	ILAVERYLKDQ

	RILAVERYLKDQ

	ARILAVERYLKDQ

	QARILAVERYLKDQ

	LQARILAVERYLKDQ

	QLQARILAVERYLKDQ

	KQLQARILAVERYLKDQ

	IKQLQARILAVERYLKDQ

	QIKQLQARILAVERYLKDQ

	WQIKQLQARILAVERYLKDQ

	VWQIKQLQARILAVERYLKDQ

	TVWQIKQLQARILAVERYLKDQ

	LTVWQIKQLQARILAVERYLKDQ

	QLTVWQIKQLQARILAVERYLKDQ

	LQLTVWQIKQLQARILAVERYLKDQ

	LLQLTVWQIKQLQARILAVERYLKDQ

	HLLQLTVWQIKQLQARILAVERYLKDQ

	QHLLQLTVWQIKQLQARILAVERYLKDQ

	QQHLLQLTVWQIKQLQARILAVERYLKDQ

	AQQHLLQLTVWQIKQLQARILAVERYLKDQ

	EAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	IEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	AIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	RAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	LRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	LLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	NLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

	NNLLRAIEAQQHLLQLTVWQIKQLQARILAVERYLKDQ

The one letter amino acid code of Table 1 is used.

TABLE 6

HIV-2_NIHZDP178 analog carboxy truncations.

	LEA

	LEAN

	LEANI

	LEANIS

	LEANISQ

	LEANISQS

	LEANISQSL

	LEANISQSLE

	LEANISQSLEQ

	LEANISQSLEQA

	LEANISQSLEQAQ

	LEANISQSLEQAQI

	LEANISQSLEQAQIQ

	LEANISQSLEQAQIQQ

	LEANISQSLEQAQIQQE

	LEANISQSLEQAQIQQEK

	LEANISQSLEQAQIQQEKN

	LEANISQSLEQAQIQQEKNM

	LEANISQSLEQAQIQQEKNMY

	LEANISQSLEQAQIQQEKNMYE

	LEANISQSLEQAQIQQEKNMYEL

	LEANISQSLEQAQIQQEKNMYELQ

	LEANISQSLEQAQIQQEKNMYELQK

	LEANISQSLEQAQIQQEKNMYELQKL

	LEANISQSLEQAQIQQEKNMYELQKLN

	LEANISQSLEQAQIQQEKNMYELQKLNS

	LEANISQSLEQAQIQQEKNMYELQKLNSW

	LEANISQSLEQAQIQQEKNMYELQKLNSWD

	LEANISQSLEQAQIQQEKNMYELQKLNSWDV

	LEANISQSLEQAQIQQEKNMYELQKLNSWDVF

	LEANISQSLEQAQIQQEKNMYELQKLNSWDVFT

	LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTN

	LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNW

	LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

The one letter amino acid code of Table 1 is used.

TABLE 7

HIV-2_NIHZDP178 analog amino truncations.

	NWL

	TNWL

	FTNWL

	VFTNWL

	DVFTNWL

	WDVFTNWL

	SWDVFTNWL

	NSWDVFTNWL

	LNSWDVFTNWL

	KLNSWDVFTNWL

	QKLNSWDVFTNWL

	LQKLNSWDVFTNWL

	ELQKLNSWDVFTNWL

	YELQKLNSWDVFTNWL

	MYELQKLNSWDVFTNWL

	NMYELQKLNSWDVFTNWL

	KNMYELQKLNSWDVFTNWL

	EKNMYELQKLNSWDVFTNWL

	QEKNMYELQKLNSWDVFTNWL

	QQEKNMYELQKLNSWDVFTNWL

	IQQEKNMYELQKLNSWDVFTNWL

	QIQQEKNMYELQKLNSWDVFTNWL

	AQIQQEKNMYELQKLNSWDVFTNWL

	QAQIQQEKNMYELQKLNSWDVFTNWL

	EQAQIQQEKNMYELQKLNSWDVFTNWL

	LEQAQIQQEKNMYELQKLNSWDVFTNWL

	SLEQAQIQQEKNMYELQKLNSWDVFTNWL

	QSLEQAQIQQEKNMYELQKLNSWDVFTNWL

	SQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

	ISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

	NISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

	ANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

	EANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

	LEANISQSLEQAQIQQEKNMYELQKLNSWDVFTNWL

The one letter amino acid code of Table 1 is used.

TABLE 8

RESPIRATORY SYNCYTIAL VIRUS (RSV) DP107 F2
REGION ANALOG CARBOXY TRUNCATIONS

YTS

YTSV

YTSVI

YTSVIT

YTSVITI

YTSVITIE

YTSVITIEL

YTSVITIELS

YTSVITIELSN

YTSVITIELSNI

YTSVITIELSNIK

YTSVITIELSNIKE

YTSVITIELSNIKEN

YTSVITIELSNIKENK

YTSVITIELSNIKENKC

YTSVITIELSNIKENKCN

YTSVITIELSNIKENKCNG

YTSVITIELSNIKENKCNGT

YTSVITIELSNIKENKCNGTD

YTSVITIELSNIKENKCNGTDA

YTSVITIELSNIKENKCNGTDAK

YTSVITIELSNIKENKCNGTDAKV

YTSVITIELSNIKENKCNGTDAKVK

YTSVITIELSNIKENKCNGTDAKVKL

YTSVITIELSNIKENKCNGTDAKVKLI

YTSVITIELSNIKENKCNGTDAKVKLIK

YTSVITIELSNIKENKCNGTDAKVKLIKQ

YTSVITIELSNIKENKCNGTDAKVKLIKQE

YTSVITIELSNIKENKCNGTDAKVKLIKQEL

YTSVITIELSNIKENKCNGTDAKVKLIKQELD

YTSVITIELSNIKENKCNGTDAKVKLIKQELDK

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKY

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYK

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKN

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNA

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAV

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVT

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTE

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTEL

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQ

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQL

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLL

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLM

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQ

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQS

YTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

The one letter amino acid code of Table 1 is used.

TABLE 9

RESPIRATORY SYNCYTIAL VIRUS (RSV) DP107 F2
REGION ANALOG AMINO TRUNCATIONS

QST

MQST

LMQST

LLMQST

QLLMQST

LQLLMQST

ELQLLMQST

TELQLLMQST

VTELQLLMQST

AVTELQLLMQST

NAVTELQLLMQST

KNAVTELQLLMQST

YKNAVTELQLLMQST

KYKNAVTELQLLMQST

DKYKNAVTELQLLMQST

LDKYKNAVTELQLLMQST

ELDKYKNAVTELQLLMQST

QELDKYKNAVTELQLLMQST

KQELDKYKNAVTELQLLMQST

IKQELDKYKNAVTELQLLMQST

LIKQELDKYKNAVTELQLLMQST

KLIKQELDKYKNAVTELQLLMQST

VKLIKQELDKYKNAVTELQLLMQST

KVKLIKQELDKYKNAVTELQLLMQST

AKVKLIKQELDKYKNAVTELQLLMQST

DAKVKLIKQELDKYKNAVTELQLLMQST

TDAKVKLIKQELDKYKNAVTELQLLMQST

GTDAKVKLIKQELDKYKNAVTELQLLMQST

NGTDAKVKLIKQELDKYKNAVTELQLLMQST

CNGTDAKVKLIKQELDKYKNAVTELQLLMQST

KCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

NKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

KENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

IKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

NIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

SNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

LSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

ELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

IELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

TIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

ITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

VITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

SVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

TSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST

The one letter amino acid code of Table 1 is used.

TABLE 10

RESPIRATORY SYNCYTIAL VIRUS (RSV) F1 DP178
REGION ANALOG CARBOXY TRUNCATIONS

	FYD

	FYDP

	FYDPL

	FYDPLV

	FYDPLVF

	FYDPLVFP

	FYDPLVFPS

	FYDPLVFPSD

	FYDPLVFPSDE

	FYDPLVFPSDEF

	FYDPLVFPSDEFD

	FYDPLVFPSDEFDA

	FYDPLVFPSDEFDAS

	FYDPLVFPSDEFDASI

	FYDPLVFPSDEFDASIS

	FYDPLVFPSDEFDASISQ

	FYDPLVFPSDEFDASISQV

	FYDPLVFPSDEFDASISQVN

	FYDPLVFPSDEFDASISQVNE

	FYDPLVFPSDEFDASISQVNEK

	FYDPLVFPSDEFDASISQVNEKI

	FYDPLVFPSDEFDASISQVNEKIN

	FYDPLVFPSDEFDASISQVNEKINQ

	FYDPLVFPSDEFDASISQVNEKINQS

	FYDPLVFPSDEFDASISQVNEKINQSL

	FYDPLVFPSDEFDASISQVNEKINQSLA

	FYDPLVFPSDEFDASISQVNEKINQSLAF

	FYDPLVFPSDEFDASISQVNEKINQSLAFI

	FYDPLVFPSDEFDASISQVNEKINQSLAFIR

	FYDPLVFPSDEFDASISQVNEKINQSLAFIRK

	FYDPLVFPSDEFDASISQVNEKINQSLAFIRKS

	FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSD

	FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDE

	FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDEL

	FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

The one letter amino acid code of Table 1 is used.

TABLE 11

RESPIRATORY SYNCYTIAL VIRUS (RSV) F1 DP178
REGION ANALOG AMINO TRUNCATIONS

	DELL

	SDELL

	KSDELL

	RKSDELL

	IRKSDELL

	FIRKSDELL

	AFIRKSDELL

	LAFIRKSDELL

	SLAFIRKSDELL

	QSLAFIRKSDELL

	NQSLAFIRKSDELL

	INQSLAFIRKSDELL

	KINQSLAFIRKSDELL

	EKINQSLAFIRKSDELL

	NEKINQSLAFIRKSDELL

	VNEKINQSLAFIRKSDELL

	QVNEKINQSLAFIRKSDELL

	SQVNEKINQSLAFIRKSDELL

	ISQVNEKINQSLAFIRKSDELL

	SISQVNEKINQSLAFIRKSDELL

	ASISQVNEKINQSLAFIRKSDELL

	DASISQVNEKINQSLAFIRKSDELL

	FDASISQVNEKINQSLAFIRKSDELL

	EFDASISQVNEKINQSLAFIRKSDELL

	DEFDASISQVNEKINQSLAFIRKSDELL

	SDEFDASISQVNEKINQSLAFIRKSDELL

	PSDEFDASISQVNEKINQSLAFIRKSDELL

	FPSDEFDASISQVNEKINQSLAFIRKSDELL

	VFPSDEFDASISQVNEKINQSLAFIRKSDELL

	LVFPSDEFDASISQVNEKINQSLAFIRKSDELL

	PLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

	DPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

	YDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

The one letter amino acid code of Table 1 is used.

TABLE 12

HUMAN PARAINFLUENZA VIRUS 3 (HPV3) F1 REGION DP178
ANALOG CARBOXY TRUNCATIONS

	ITL

	ITLN

	ITLNN

	ITLNNS

	ITLNNSV

	ITLNNSVA

	ITLNNSVAL

	ITLNNSVALD

	ITLNNSVALDP

	ITLNNSVALDPI

	ITLNNSVALDPID

	ITLNNSVALDPIDI

	ITLNNSVALDPIDIS

	ITLNNSVALDPIDISI

	ITLNNSVALDPIDISIE

	ITLNNSVALDPIDISIEL

	ITLNNSVALDPIDISIELN

	ITLNNSVALDPIDISIELNK

	ITLNNSVALDPIDISIELNKA

	ITLNNSVALDPIDISIELNKAK

	ITLNNSVALDPIDISIELNKAKS

	ITLNNSVALDPIDISIELNKAKSD

	ITLNNSVALDPIDISIELNKAKSDL

	ITLNNSVALDPIDISIELNKAKSDLE

	ITLNNSVALDPIDISIELNKAKSDLEE

	ITLNNSVALDPIDISIELNKAKSDLEES

	ITLNNSVALDPIDISIELNKAKSDLEESK

	ITLNNSVALDPIDISIELNKAKSDLEESKE

	ITLNNSVALDPIDISIELNKAKSDLEESKEW

	ITLNNSVALDPIDISIELNKAKSDLEESKEWI

	ITLNNSVALDPIDISIELNKAKSDLEESKEWIR

	ITLNNSVALDPIDISIELNKAKSDLEESKEWIRR

	ITLNNSVALDPIDISIELNKAKSDLEESKEWIRRS

The one letter amino acid code of Table 1 is used.

TABLE 13

HUMAN PARAINFLUENZA VIRUS 3 (HPV3) F1 REGION DP178
ANALOG AMINO TRUNCATIONS

	RRS

	IRRS

	WIRRS

	EWIRRS

	KEWIRRS

	SKEWIRRS

	ESKEWIRRS

	EESKEWIRRS

	LEESKEWIRRS

	DLEESKEWIRRS

	SDLEESKEWIRRS

	KSDLEESKEWIRRS

	AKSDLEESKEWIRRS

	KAKSDLEESKEWIRRS

	NKAKSDLEESKEWIRRS

	LNKAKSDLEESKEWIRRS

	ELNKAKSDLEESKEWIRRS

	IELNKAKSDLEESKEWIRRS

	SIELNKAKSDLEESKEWIRRS

	ISIELNKAKSDLEESKEWIRRS

	DISIELNKAKSDLEESKEWIRRS

	IDISIELNKAKSDLEESKEWIRRS

	PIDISIELNKAKSDLEESKEWIRRS

	DPIDISIELNKAKSDLEESKEWIRRS

	LDPIDISIELNKAKSDLEESKEWIRRS

	ALDPIDISLELNKAKSDLEESKEWIRRS

	VALDPIDISIELNKAKSDLEESKEWIRRS

	SVALDPIDISIELNKAKSDLEESKEWIRRS

	NSVALDPIDISIELNKAKSDLEESKEWIRRS

	NNSVALDPIDISIELNKAKSDLEESKEWIRRS

	LNNSVALDPIDISIELNKAKSDLEESKEWIRRS

	TLNNSVALDPIDISIELNKAKSDLEESKEWIRRS

The one letter amino acid code of Table 1 is used.

TABLE 14

HUMAN PARAINFLUENZA VIRUS 3 (HPV3) F1 REGION
DP107 ANALOG CARBOXY TRUNCATIONS

	ALG

	ALGV

	ALGVA

	ALGVAT

	ALGVATS

	ALGVATSA

	ALGVATSAQ

	ALGVATSAQI

	ALGVATSAQIT

	ALGVATSAQITA

	ALGVATSAQITAA

	ALGVATSAQITAAV

	ALGVATSAQITAAVA

	ALGVATSAQITAAVAL

	ALGVATSAQITAAVALV

	ALGVATSAQITAAVALVE

	ALGVATSAQITAAVALVEA

	ALGVATSAQITAAVALVEAK

	ALGVATSAQITAAVALVEAKQ

	ALGVATSAQITAAVALVEAKQA

	ALGVATSAQITAAVALVEAKQAR

	ALGVATSAQITAAVALVEAKQARS

	ALGVATSAQITAAVALVEAKQARSD

	ALGVATSAQITAAVALVEAKQARSDI

	ALGVATSAQITAAVALVEAKQARSDIE

	ALGVATSAQITAAVALVEAKQARSDIEK

	ALGVATSAQITAAVALVEAKQARSDIEKL

	ALGVATSAQITAAVALVEAKQARSDIEKLK

	ALGVATSAQITAAVALVEAKQARSDIEKLKE

	ALGVATSAQITAAVALVEAKQARSDIEKLKEA

	ALGVATSAQITAAVALVEAKQARSDIEKLKEAI

	ALGVATSAQITAAVALVEAKQARSDIEKLKEAIR

The one letter amino acid code of Table 1 is used.

TABLE 15

HUMAN PARAINFLUENZA VIRUS 3 (HPV3) F1 REGION
DP107 ANALOG AMINO TRUNCATIONS

	IRD

	AIRD

	EAIRD

	KEAIRD

	LKEAIRD

	KLKEAIRD

	EKLKEAIRD

	IEKLKEAIRD

	DIEKLKEAIRD

	SDIEKLKEAIRD

	RSDIEKLKEAIRD

	ARSDIEKLKEAIRD

	QARSDIEKLKEAIRD

	KQARSDIEKLKEAIRD

	AKQARSDIEKLKEAIRD

	EAKQARSDIEKLKEAIRD

	VEAKQARSDIEKLKEAIRD

	LVEAKQARSDIEKIKEAIRD

	ALVEAKQARSDIEKLKEAIRD

	VALVEAKQARSDIEKLKEAIRD

	AVALVEAKQARSDIEKLKEAIRD

	AAVALVEAKQARSDIEKLKEAIRD

	TAAVALVEAKQARSDIEKLKEAIRD

	ITAAVALVEAKQARSDIEKLKEAIRD

	QITAAVALVEAKQARSDIEKLKEAIRD

	AQITAAVALVEAKQARSDIEKLKEAIRD

	SAQITAAVALVEAKQARSDIEKLKEAIRD

	TSAQITAAVALVEAKQARSDIEKLKEAIRD

	ATSAQITAAVALVEAKQARSDLEKLKEAIRD

	VATSAQITAAVALVEAKQARSDIEKLKEAIRD

	GVATSAQITAAVALVEAKQARSDIEKLKEAIRD

	LGVATSAQITAAVALVEAKQARSDIEKLKEAIRD

The one letter amino acid code of Table 1 is used.

TABLE 16

ANTI-RESPIRATORY SYNCYTIAL VIRUS (RSV) PEPTIDES

	TSVITIELSNIKENKCNGTDAKVKLIKQELDKYKN

	SVITIELSNIKENKCNGTDAKVKLIKQELDKYKNA

	VITIELSNIKENKCNGTDAKVKLIKQELDKYKNAV

	VAVSKVLHLEGEVNKIALLSTNKAVVSLSNGVS

	AVSKVLHLEGEVNKIALLSTNKAVVSLSNGVSV

	VSKVLHLEGEVNKIALLSTNKAVVSLSNGVSVL

	SKVLHLEGEVNKIALLSTNKAVVSLSNGVSVLT

	KVLHLEGEVNKIALLSTNKAVVSLSNGVSVLTS

	LEGEVNKIALLSTNKAVVSLSNGVSVLTSKVLD

	GEVNKIALLSTNKAVVSLSNGVSVLTSKVLDLK

	EVNKIALLSTNKAVVSLSNGVSVLTSKVLDLKN

	VNKIALLSTNKAVVSLSNGVSVLTSKVLDLKNY

	NKIALLSTNKAVVSLSNGVSVLTSKVLDLKNYI

	KIALLSTNKAVVSLSNGVSVLTSKVLDLKNYID

	IALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK

	ALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ

	VAVSKVLHLEGEVNKIALLSTNKAVVSLSNGVS

	AVSKVLHLEGEVNKIALLSTNKAVVSLSNGVSV

	VSKVLHLEGEVNKIALLSTNKAVVSLSNGVSVL

	SKVLHLEGEVNKIALLSTNKAVVSLSNGVSVLT

	KVLHLEGEVNKIALLSTNKAVVSLSNGVSVLTS

	LEGEVNKIALLSTNKAVVSLSNGVSVLTSKVLD

	GEVNKIALLSTNKAVVSLSNGVSVLTSKVLDLK

	EVNKIALLSTNKAVVSLSNGVSVLTSKVLDLKN

	VNKIALLSTNKAVVSLSNGVSVLTSKVLDLKNY

	NKIALLSTNKAVVSLSNGVSVLTSKVLDLKNYI

	KIALLSTNKAVVSLSNGVSVLTSKVLDLKNYID

	IALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDK

	ALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ

The one letter amino acid code of Table 1 is used.

TABLE 17

ANTI-HUMAN PARAINFLUENZA VIRUS 3 (HPV3) PEPTIDES

	TLNNSVALDPIDISIELNKAKSDLEESKEWIRRSN

	LNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQ

	NNSVALDPIDISIELNKAKSDLEESKEWIRRSNQK

	NSVALDPIDISIELNKAKSDLEESKEWIRRSNQKL

	SVALDPIDISIELNKAKSDLEESKEWIRRSNQKLD

	VALDPIDISIELNKAKSDLEESKEWIRRSNQKLDS

	ALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSI

	LDPIDISIELNKAKSDLEESKEWIRRSNQKLDSIG

	DPIDISIELNKAKSDLEESKEWIRRSNQKLDSIGN

	PIDISIELNKAKSDLEESKEWIRRSNQKLDSIGNW

	IDISIELNKAKSDLEESKEWIRRSNQKLDSIGNWH

	DISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQ

	ISIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQS

	SIELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSS

	IELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSST

	ELNKAKSDLEESKEWIRRSNQKLDSIGNWHQSSTT

	TAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQS

	AVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSI

	LVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNL

	VEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLI

	EAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIV

	AKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVA

	KQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAI

	QARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIK

	ARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKS

	RSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSV

	SDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQ

	KLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVN

	LKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNK

	AIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIV

The one letter amino acid code of Table 1 is used.

TABLE 18

ANTI-SIMIAN IMMUNODEFICIENCY VIRUS (SIV) PEPTIDES

	WQEWERKVDFLEENITALLEEAQIQQEKNMYELQK

	QEWERKVDFLEENITALLEEAQIQQEKNMYELQKL

	EWERKVDFLEENITALLEEAQIQQEKNMYELQKLN

	WERKVDFLEENITALLEEAQIQQEKNMYELQKLNS

	ERKVDFLEENITALLEEAQIQQEKNMYELQKLNSW

	RKVDFLEENITALLEEAQIQQEKNMYELQKLNSWD

	KVDFLEENITALLEEAQIQQEKNMYELQKLNSWDV

	VDFLEENITALLEEAQIQQEKNMYELQKLNSWDVF

	DFLEENITALLEEAQIQQEKNMYELQKLNSWDVFG

	FLEENITALLEEAQIQQEKNMYELQKLNSWDVFGN

The one letter amino acid code of Table 1 is used.

TABLE 19

ANTI-MEASLES VIRUS (MEV) PEPTIDES

	LHRIDLGPPISLERLDVGTNLGNAIAKLEAKELL

	HRIDLGPPISLERLDVGTNLGNAIAKLEAKELLE

	RIDLGPPISLERLDVGTNLGNAIAKLEAKELLES

	IDLGPPISLERLDVGTNLGNAIAKLEAKELLESS

	DLGPPISLERLDVGTNLGNAIAKLEAKELLESSD

	LGPPISLERLDVGTNLGNAIAKLEAKELLESSDQ

	GPPISLERLDVGTNLGNAIAKLEAKELLESSDQI

	PPISLERLDVGTNLGNAIAKLEAKELLESSDQIL

	PISLERLDVGTNLGNAIAKLEAKELLESSDQILR

	SLERLDVGTNLGNAIAKLEAKELLESSDQILRSM

	LERLDVGTNLGNAIAKLEAKELLESSDQILRSMK

The one letter amino acid code of Table 1 is used.

Claims

1. A modified anti-viral peptide comprising:

a peptide that exhibits anti-viral activity against respiratory syncytial virus (RSV), and

a reactive group which is reactive with amino groups, hydroxyl groups, or thiol groups on blood components to form stable covalent bonds.

2. The modified peptide of claim 1, wherein said reactive group is a succinimidyl or a maleimido group.

3. The modified peptide of claim 1, wherein said reactive group is a maleimido group which is reactive with a thiol group on a blood protein.

4.-8. (canceled)

9. The modified peptide of claim 1, wherein said peptide is selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 30.

10. The modified peptide of claim 1, wherein said peptide is selected from the group consisting of SEQ ID N0: 14 to SEQ ID N0: 17 and SEQ ID NO: 29.

11. A modified anti-viral peptide comprising:

a peptide exhibits antiviral activity against human parainfluenza virus (HPIV), and

12. The modified peptide of claim 11, wherein said peptide is selected from the group consisting of SEQ ID N0: 31 to SEQ ID N0: 62.

13. The modified peptide of claim 11, wherein said peptide is selected from the group consisting of SEQ ID NO: 35, SEQ ID N0: 38 to SEQ ID N0: 42, SEQ ID N0: 52 and SEQ ID N0: 58.

14. A modified anti-viral peptide comprising:

a peptide exhibits antiviral activity against measles virus (MeV), and

15. The modified peptide of claim 14, wherein said peptide is selected from the group consisting of SEQ ID N0: 74 to SEQ ID N0: 86.

16. The modified peptide of claim 14, wherein said peptide is selected from the group consisting of SEQ ID N0: 77, SEQ ID N0: 79, SEQ ID N0: 81 and SEQ ID N0: 84.

17. A modified anti-viral peptide comprising:

a peptide exhibits antiviral activity against simian immunodeficiency virus (SIV), and

18. The modified peptide of claim 17, wherein said peptide is selected from the group consisting of SEQ ID N0: 63 to SEQ ID N0: 73.

19. (canceled)

20. The modified peptide of claim 11, wherein said reactive group is a maleimido group which is reactive with a thiol group on a blood protein.

21.-22. (canceled)

23. The modified peptide of claim 14, wherein said reactive group is a maleimido group which is reactive with a thiol group on a blood protein.

24.-25. (canceled)

26. The modified peptide of claim 17, wherein said reactive group is a maleimido group which is reactive with a thiol group on a blood protein.

27.-30. (canceled)

31. The modified peptide of claim 11, wherein said reactive group is a succinimidyl or a maleimido group.

32. The modified peptide of claim 14, wherein said reactive group is a succinimidyl or a maleimido group.

33. The modified peptide of claim 17, wherein said reactive group is a succinimidyl or a maleimido group.

34. An anti-viral peptide-albumin conjugate comprising:

a peptide that exhibits anti-viral activity against respiratory syncytial virus (RSV) that comprises an amino acid sequence and a reactive group which is reactive with amino groups, hydroxyl groups, or thiol groups on blood components to form stable covalent bonds; and

albumin, wherein the peptide is covalently bonded to the albumin through the reactive group.

35. The conjugate of claim 34, wherein said reactive group is a maleimido group which is reactive with a thiol group on the albumin.

36. The conjugate of claim 34, wherein said peptide is selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 30.

37. The conjugate of claim 34, wherein said peptide is selected from the group consisting of SEQ ID N0: 14 to SEQ ID N0: 17 and SEQ ID NO: 29.

38. An anti-viral peptide-albumin conjugate comprising:

a peptide that exhibits anti-viral activity against human parainfluenza virus (HPIV), that comprises an amino acid sequence and a reactive group which is reactive with amino groups, hydroxyl groups, or thiol groups on blood components to form stable covalent bonds; and

39. The conjugate of claim 38, wherein said reactive group is a maleimido group which is reactive with a thiol group on the albumin.

40. The conjugate of claim 38, wherein said peptide is selected from the group consisting of SEQ ID N0: 31 to SEQ ID N0: 62.

41. The conjugate of claim 38, wherein said peptide is selected from the group consisting of SEQ ID N0: 35, SEQ ID N0: 38 to 42, SEQ ID N0: 52 and SEQ ID N0: 58.

42. An anti-viral peptide-albumin conjugate comprising:

a peptide that exhibits anti-viral activity against measles virus (MeV), that comprises an amino acid sequence and a reactive group which is reactive with amino groups, hydroxyl groups, or thiol groups on blood components to form stable covalent bonds; and

43. The conjugate of claim 42, wherein said reactive group is a maleimido group which is reactive with a thiol group on the albumin.

44. The conjugate of claim 42, wherein said peptide is selected from the group consisting of SEQ ID N0: 74 to SEQ ID N0: 86.

45. The conjugate of claim 42 wherein said peptide is selected from the group consisting of SEQ ID N0: 77, SEQ ID N0: 79, SEQ ID N0: 81 and SEQ ID N0: 84.

46. An anti-viral peptide-albumin conjugate comprising:

a peptide that exhibits anti-viral activity against simian immunodeficiency virus (SIV), that comprises an amino acid sequence and a reactive group which is reactive with amino groups, hydroxyl groups, or thiol groups on blood components to form stable covalent bonds; and

47. The conjugate of claim 46, wherein said reactive group is a maleimido group which is reactive with a thiol group on the albumin.

48. The conjugate of claim 46, wherein said peptide is selected from the group consisting of SEQ ID N0: 63 to SEQ ID N0: 73.

49. A composition comprising the modified anti-viral peptide of claim 1 and a physiologically acceptable medium.

50. A composition comprising the modified anti-viral peptide of claim 11 and a physiologically acceptable medium.

51. A composition comprising the modified anti-viral peptide of claim 14 and a physiologically acceptable medium.

52. A composition comprising the modified anti-viral peptide of claim 17 and a physiologically acceptable medium.

53. A composition comprising the conjugate of claim 34 and a physiologically acceptable medium.

54. A composition comprising the conjugate of claim 38 and a physiologically acceptable medium.

55. A composition comprising the conjugate of claim 42 and a physiologically acceptable medium.

56. A composition comprising the conjugate of claim 46 and a physiologically acceptable medium.

57. A method of inhibiting or reducing membrane fusion between respiratory syncytial virus (RSV) and a cell, comprising contacting the RSV with a modified peptide of claim 1.

58. A method of inhibiting or reducing membrane fusion between human parainfluenze virus (HPIV) and a cell, comprising contacting the HPIV with a modified peptide of claim 11.

59. A method of inhibiting or reducing membrane fusion between measles virus (MeV) and a cell, comprising contacting the MeV with a modified peptide of claim 14.

60. A method of inhibiting or reducing membrane fusion between simian immunodeficiency virus (SIV) and a cell, comprising contacting the SIV with a modified peptide of claim 17.

61. A method of inhibiting or reducing membrane fusion between respiratory syncytial virus (RSV) and a cell, comprising contacting the RSV with a conjugate of claim 34.

62. A method of inhibiting or reducing membrane fusion between human parainfluenze virus (HPIV) and a cell, comprising contacting the HPIV with a conjugate of claim 38.

63. A method of inhibiting or reducing membrane fusion between measles virus (MeV) and a cell, comprising contacting the MeV with a conjugate of claim 42.

64. A method of inhibiting or reducing membrane fusion between simian immunodeficiency virus (SIV) and a cell, comprising contacting the SIV with a conjugate of claim 46.

65. A method of making a conjugate of claim 34, comprising:

contacting a modified peptide of claim 1 with albumin to form an anti-viral peptide-albumin conjugate.

66. The method of claim 65, wherein the modified anti-viral peptide is made by combining an anti-RSV peptide having a free amino group, N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) and trietylamine or maleimidopropionic acid (MPA) to form a maleimide containing group at the free amino group, to form the modified anti-viral peptide.

67. A method of making a conjugate of claim 38, comprising:

contacting a modified peptide of claim 11 with albumin to form an anti-viral peptide-albumin conjugate.

68. The method of claim 67, wherein the modified anti-viral peptide is made by combining an anti-HPIV peptide having a free amino group, N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) and trietylamine or maleimidopropionic acid (MPA) to form a maleimide containing group at the free amino group, to form the modified anti-viral peptide.

69. A method of making a conjugate of claim 42, comprising:

contacting a modified peptide of claim 14 with albumin to form an anti-viral peptide-albumin conjugate.

70. The method of claim 69, wherein the modified anti-viral peptide is made by combining an anti-MeV peptide having a free amino group, N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) and trietylamine or maleimidopropionic acid (MPA) to form a maleimide containing group at the free amino group, to form the modified anti-viral peptide.

71. A method of making a conjugate of claim 46, comprising:

contacting a modified peptide of claim 17 with albumin to form an anti-viral peptide-albumin conjugate.

72. The method of claim 71, wherein the modified anti-viral peptide is made by combining an anti-SIV peptide having a free amino group, N-[γ-maleimidobutyryloxy]succinimide ester (GMBS) and trietylamine or maleimidopropionic acid (MPA) to form a maleimide containing group at the free amino group, to form the modified anti-viral peptide.

73. A method of treating or preventing respiratory syncytial virus (RSV) infection in a subject, comprising

administering a modified peptide of claim 1 to a subject having or at risk of RSV infection, thereby treating or preventing the infection.

74. The method of claim 73, wherein the subject has brochiolitis.

75. The method of claim 73, wherein the subject has pneumonia.

76. The method of claim 73, wherein the modified peptide is conjugated with albumin in vivo.

77. The method of claim 73, wherein the modified peptide is conjugated with albumin ex vivo.

78. A method of treating or preventing respiratory syncytial virus (RSV) infection in a subject, comprising

administering a conjugate of claim 34 to a subject having or at risk of RSV infection, thereby treating or preventing the infection.

79. The method of claim 78, wherein the subject has brochiolitis.

80. The method of claim 78, wherein the subject has pneumonia.

81. A method of treating or preventing human parainfluenza virus (HPIV) infection in a subject, comprising

administering a modified peptide of claim 11 to a subject having or at risk of HPIV infection, thereby treating or preventing the infection.

82. The method of claim 81, wherein the subject has respiratory tract disease.

83. The method of claim 81, wherein the subject has croup.

84. The method of claim 81, wherein the subject has brochiolitis.

85. The method of claim 81, wherein the subject has pneumonia.

86. The method of claim 81, wherein the modified peptide is conjugated with albumin in vivo.

87. The method of claim 81, wherein the modified peptide is conjugated with albumin ex vivo.

88. A method of treating or preventing human parainfluenza virus (HPIV) infection in a subject, comprising

administering a conjugate of claim 38 to a subject having or at risk of HPIV infection, thereby treating or preventing the infection.

89. The method of claim 88, wherein the subject has respiratory tract disease.

90. The method of claim 88, wherein the subject has croup.

91. The method of claim 88, wherein the subject has brochiolitis.

92. The method of claim 88, wherein the subject has pneumonia.

93. A method of treating or preventing measles virus (MeV) infection in a subject, comprising

administering a modified peptide of claim 14 to a subject having or at risk of MeV infection, thereby treating or preventing the infection.

94. The method of claim 93, wherein the modified peptide is conjugated with albumin in vivo.

95. The method of claim 93, wherein the modified peptide is conjugated with albumin ex vivo.

96. A method of treating or preventing measles virus (MeV) infection in a subject, comprising

administering a conjugate of claim 42 to a subject having or at risk of MeV infection, thereby treating or preventing the infection.

97. A method of treating or preventing simian immunodeficiency virus (SIV) infection in a subject, comprising

administering a modified peptide of claim 17 to a subject having or at risk of SIV infection, thereby treating or preventing the infection.

98. The method of claim 97, wherein the modified peptide is conjugated with albumin in vivo.

99. The method of claim 97, wherein the modified peptide is conjugated with albumin ex vivo.

100. A method of treating or preventing simian immunodeficiency virus (SIV) infection in a subject, comprising

administering a conjugate of claim 46 to a subject having or at risk of SIV infection, thereby treating or preventing the infection.