J. Peptide Protein Res. 47, 1996, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Int. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`0 Munksgaard zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Printed in Belgium zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Copjrighf zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`ISSN 03674377 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`1996
`INTERNATIONAL JOURNAL OF
`PEPTIDE & PROTEIN RESEARCH
`
`YI-97
`- all rights reserved
`
`Automated multiple peptide synthesis: Improvements in obtaining
`quality peptides
`
`THONG LUU, SON PHAM and SHRIKANT DESHPANDE
`
`Anergen Inc., Redwood City, California, USA
`
`Received 9 May, revised 20 June, accepted for publication 20 August 1995
`
`Production of multiple overlapping peptides is a key step in the identification of T-cell epitopes. A large number
`of peptides can be produced by using ABIMED’s automated multiple peptide synthesizer. We report here
`considerable improvement in the software and chemistry of peptide synthesis by introducing a resin mixing
`step during coupling, when using this synthesizer. A comparison of two solvent systems for synthesis was
`performed. Six test peptides were synthesized by standard and modified methods. The purity of peptides,
`assessed by HPLC and mass spectrometry, showed a substantial improvement when automated resin mix-
`ing and mixed solvent system were used. These improvements enable us to produce 48 peptides within a week
`each of sufficient purity to be used for rapid screening of T-cell epitopes. 0 Munksgaard 1996.
`
`Key words: coupling solvent; multiple peptide synthesis; resin mixing; T-cell epitopes
`
`Solid-phase peptide synthesis (SPPS), invented by
`Merrifield (l), involves three distinct steps: (1) chain
`assembly on the solid phase; (2)cleavage of peptide
`from the solid support along with deprotection of side
`chains of amino acid residues; (3) purification and char-
`acterization of peptides. In the preparation of a large
`number of peptides for T cell epitope screening, the
`purification step is time consuming and not practical.
`Ideally, for the production of multiple peptides, the
`cleaved crude peptides should be sufficiently pure that
`they can be directly used in T cell epitope screening.
`Several automated and semi-automated multiple pep-
`tide synthesizers (MPS) are available commercially
`(2-6) which can be used to prepare multiple peptides.
`However, to achieve the goal of producing pure pep-
`tides of more than 12 amino acids, several modifica-
`tions have to be made such as the use of superior cou-
`pling agents, multiple couplings of Na-blocked amino
`
`AA, amino acid; Ac, acetyl; Ac-DR4DW4p57-76, acetyl-human
`major histocompatibility complex DR4DW4 P-chain sequence 57-
`76; BOP, benzotriazol-l-yloxytr~s(dimethy1amino)phosphonium
`hexafluorophosphonate; CDI, carbonyldiimidazole; DCM, dichloro-
`methane; DIEA, diisopropylethylamine; DMF, dimethylformamide;
`1,1,3,3-tetramethyluroniumhexa-
`fluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt,
`1 -hydroxybenzotriazole; MBP,m yelin basic protein; NMM, N-methyl-
`morpholine; PyBOP, benzotriazole-1-yloxytris(pyrro1idino)phos-
`phonium hexafluorophosphate; GP, guinea pig.
`
`acids or transfer to a better solvent system. Sometimes
`one or all these modifications has to be introduced in
`order to get optimum peptide purity.
`We were interested in the preparation of 14-20-mer
`overlapping peptides of antigenic proteins invoked in
`the autoimmune diseases, viz. multiple sclerosis (MS),
`myasthenia gravis (MG), rheumatoid arthritis (RA) and
`insulin-dependent diabetes mellitus (IDDM). We em-
`ployed an ABIMED-Gilson AMS 422 MPS for this
`purpose. However, the peptides synthesized by PyBOP
`coupling chemistry in dimethylformamide (DMF) using
`standard protocols provided by ABIMED did not pro-
`duce the desired level of purity. There were several
`problems in using the standard protocol. First, with no
`mixing of resin during coupling of amino acids to the
`growing chain on the resin, the polystyrene resin used
`as solid support settled at the bottom of the column,
`thus decreasing the efficiency of aminoacylation, as well
`as Fmoc deprotection. Second, although D M F is con-
`sidered to be a good solvent for peptide synthesis, it is
`not an ideal solvent for the preparation of longer pep-
`tides in which growing peptide chains tend to form a
`b-sheet structure (7). Efforts to eliminate B-sheet struc-
`tures by the addition of more polar solvents such as
`trifluoroethanol (TFE) and hexafluoroisopropanol have
`been successful (8,9). A problem that is usually ignored
`is that in cases when aggregation occurs due to apolar
`side-chain protecting groups, increased solvent polarity
`does not help to disrupt the aggregation and a nonpolar
`
`91
`
`HBTU, 2-( 1H-benzotriazol- 1-y1)- zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`MPI EXHIBIT 1037 PAGE 1
`
`MPI EXHIBIT 1037 PAGE 1
`
`

`

`T. Luu zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`T.4BLE zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Sequerices zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`of peptides ,jxrhesi:ed zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Ac-YDENPVVHFFKNIVTPRTPP-NH2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`GP-MBP69-88 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`et al.
`
`1
`ori A M S 4 2 2 imlriple pepride syrirhesizer
`
`Peptide name
`
`Sequence
`
`~ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Ac-MBP83- 102Y83
`Ac-DR3DW4b57-76
`
`Peptide
`no.
`
`~
`
`1
`2
`3
`4
`5
`6
`
`Ac-MBPl-14
`RatMBP87-99
`Collagen Type I1 274-288
`
`Ac-DAEYWNSQKDLLEQRAAVD-NH2
`H-GSLPQKSQRSQDENPVVHF-NHz
`Ac-ASQKRPSQRHGSKY-NHz
`H-VHFFKNIVTPRTP-NH2
`H-GI AGFKGEQGPKGEP-NHz
`
`solvent may be necessary. These problems require im-
`provements in both robotics and chemistry of coupling
`to suppress deletion or addition-peptide formation and
`obtain high-purity peptides. To resolve these technical
`hurdles, six test peptide fragments from different anti-
`genic proteins associated with autoimmune disease
`were prepared by standard, and several modifications
`of standard, coupling procedures. The purity of these
`peptides were compared.
`
`EXPERIMENTAL PROCEDURES
`
`Peptide synthesis
`Six peptides with sequences (Table 1) were synthesized
`on a 0.25 mmol scale using an ABI 43 1A peptide syn-
`thesizer by FastMoc chemistry (10). HBTU/HOBt ac-
`tivation of Nz-protected amino acids was employed for
`coupling. The side-chain protecting groups used in all
`the syntheses are given in Table 2. Rink amide-MBHA
`(Novabiochem, San Diego; substitution: 0.55 mmoLg)
`resin was used for all syntheses. The peptides were
`trifluoroacetic acid containing 4-
`cleaved using
`methoxybenzenethiol and 4-(methy1mercapto)phenol as
`scavengers. The crude peptides were precipitated in
`pentane:acetone (4: 1). They were purified by prepara-
`tive reversed-phase high-performance liquid chromato-
`0, aqueous aceto-
`nitrile containing 0.1% TFA gradient. The purity of
`each final product was assessed by analytical RP-
`HPLC, and the peptides were characterized by fast
`
`TABLE 2
`Side-chain protecting groups used in the pepride rj~rithesis
`
`Amino acid
`
`Side-cham protecting group
`
`S, T, Y, D and E
`C, N, Q and H
`R
`
`K and W
`
`92
`
`rert-Butyl (t-Bu)
`Tntyl (Trt)
`2,2,5,7,8-Pentaniethj Ichloraman-h-
`sulfonql (Pmc)
`Butyloxqcarbonjl (Boc)
`
`atom bombardment mass spectrometry (FAB-MS).
`These peptides were used as standards for coelution
`studies for the peptides obtained with the multiple pep-
`tide synthesizer.
`
`Peptide synthesis on a multiple peptide synthesizer
`Our ABIMED-Gilson AMS 422 multiple peptide syn-
`thesizer was obtained from Gilson. The synthesizer
`consists of a Gilson auto-sampler which is capable of
`X - Y - 2 movements, a 48-column reactor module and
`amino acid and activating reagent reservoirs. While the
`reagents and solvents were added to each column by a
`micro-injector sequentially, the washing of resin in all
`reaction columns was performed simultaneously.
`
`Multiple peptide sjwthesis
`
`Method A : multiple peptide synthesis in DMF by standard
`protocol without resin mixing. The six peptides (Table 1)
`were synthesized on MPS-AMS 422 using PyBOP
`chemistry following a standard protocol suggested by
`ABIMED
`(Fig. la). Rink
`amide-MBHA
`resin
`(0.025 mmol of active sites per reaction column) was
`used for synthesis. The coupling reactions were per-
`formed in DMF. Deprotection of Fmoc groups was
`carried out in 20% piperidine in D M F for 6.5 min at
`the start of the synthesis and gradually increased to
`19.5 min when amino acid chain length on the resin was
`increased to 20. A double deprotection strategy was
`used at each step to ensure complete deprotection of
`Fmoc groups before the next amino acid coupling. The
`coupling of amino acids was achieved by using a molar
`ratio of active sites on the coupling resin to Fmoc-
`amino acid as 1:6. The amino acids were double-
`coupled to ensure the completion of reaction.
`
`Method B: nzultiple peptide synthesis by mixing the resin
`in DMF. The six peptides were prepared on MPS-AMS-
`422 by the protocol given in Fig. 1. The software on the
`computer was modified to accommodate resin mixing
`after the coupling solutions were delivered to each re-
`action column. The resin mixing was performed for 35 s
`by gently bubbling nitrogen through the reaction col-
`
`graphy (RH-HPLC), using 0-70 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`MPI EXHIBIT 1037 PAGE 2
`
`MPI EXHIBIT 1037 PAGE 2
`
`

`

`Method A
`
`Method B
`
`Resin
`
`Fmoc removal (2 times)
`
`Resin Mixing
`
`Wash resin
`
`AAI coupling 1
`
`J-
`1
`1
`J. E E mixing
`Wash resin I
`J- E E mixing
`I
`
`AA1 coupling 2
`
`Fmoc removal
`
`AAn coupling
`
`Fmoc removal (2 times)
`No resin mixing
`
`1
`J.
`Wash resin I
`+
`
`AAlcouplingl
`I
`DMF
`NO resin mixing
`
`Wash resin
`
`AAl coupling 2
`
`1 E E e s i n mixing
`I Fmoc removal
`
`AAn coupling
`
`J.
`
`Method C
`
`Resin
`
`Fmoc removal (2 times)
`
`Resin Mixing
`
`Wash resin
`
`1
`1
`J.
`
`AA1 coupling 1
`DMF+DCM (3:l)
`Resin mixing
`
`Wash resin
`
`AA1 coupling 2
`DMF+DCM (3:l)
`Resin mixing
`
`1
`I +
`+
`
`Fmoc removal
`
`AAn coupling
`
`J.
`
`Fmoc removal
`DMF, DCM wash
`Synthesis Complete
`
`Automated multiple peptide synthesis zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Resin zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`+ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`Cleavage and isolation zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`tion column. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Method zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`peptides were also prepared on MPS AMS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`Fmoc removal
`DMF,DCMwash
`Synthesis Complete
`
`Fmoc removal
`DMF, DCM wash
`Synthesis Complete
`
`FIGURE 1
`The flow chart of three methods used for the synthesis of peptides on AMS 422 multiple peptide synthesizer. Deprotection was carried out
`for 6.5 min for the first amino acid and it was linearly increased at each cycle to achieve a deporotection time of 19.5 min at cycle 20. A
`similar strategy was used for coupling time with basic coupling time of 30 min for the first cycle, increasing to 90 min the for 20th cycle.
`
`umn. Each delivery of coupling solution and deprotec-
`tion solution was repeated three times for each column
`before delivering the next amino acid to another reac-
`
`C: multiple peptide synthesis by improved proto-
`col (resin mixing and DMF-DCM mixed solvent). The six
`422 by the
`new protocol as given in Fig. 1. The method was similar
`to Method B described above except one minute after
`Fmoc-amino acid, PyBOP, HOBt and NMM were de-
`livered to the reaction column, DCM was delivered to
`it. The ratio of DCM:DMF (v:v) in the reaction mix-
`ture was 1:3. Resin mixing was performed similarly to
`Method B after the DCM was delivered.
`
`of crude peptides synthesized on
`multiple peptide synthesizer by standard or improved
`protocol
`After the final Fmoc deprotection, the peptide resins
`were washed with DCM and dried in the reaction col-
`umns by applying vacuum on the synthesizer. Columns
`were removed from the synthesizer and capped at one
`end using syringe caps (Gilson part # 3980025). TFA
`(1.5 mL), containing 0.07 g of 4-(methy1mercapto)-
`phenol, and 0.1 mL of 4-methoxybenzenethiol, was
`added to each column followed by mixing at room tem-
`perature (r.t.) for 2 h. Upon completion of cleavage, the
`caps were replaced by PTFE minifilters (Nalgene part
`# 199-2045). The reaction mixture was filtered and the
`filtrate was collected into 100 mL of pentane:acetone
`(4:l). The peptides were allowed to precipitate for 2 h
`
`93
`
`MPI EXHIBIT 1037 PAGE 3
`
`MPI EXHIBIT 1037 PAGE 3
`
`

`

`al.
`
`at room temperature and isolated by centrifugation.
`They were washed three times with pentane:acetone
`and twice with pentane. The crude peptides were dried
`in vacuum for 2 h then subjected to analytical RP-
`HPLC and mass spectrometry. HPLC coelution studies
`of these peptides were performed using purified refer-
`ence standards.
`
`RESULTS AND DISCUSSION
`
`Six model peptides were designed to optimize multiple
`peptide synthesis techniques. By using resin mixing and
`two different solvent systems, peptides ranging from
`14-20 amino acids can be produced in high purity.
`These peptides were chosen based on the fact that they
`contain difficult sequences. The problems in the syn-
`
`T. Luu zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Method zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`A
`
`Peptide 1:
`
`Method B
`
`Method C
`
`Peptide 2:
`
`Peptide 3:
`
`94
`
`Fig. 2a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`MPI EXHIBIT 1037 PAGE 4
`
`MPI EXHIBIT 1037 PAGE 4
`
`

`

`Automated multiple peptide synthesis
`
`thesis of these T-cell epitopes were well documented in
`our laboratory. The three protocols which were used in
`synthesis of the six peptides on MPS are given in Fig. 1.
`Reversed-phase HPLC traces of peptides prepared by
`these protocols are given in Figs. 2a and 2b.
`The HPLC results of crude peptides prepared by
`these three methods indicated that the purity of pep-
`
`tides increased substantially by using a resin mixing
`A) gave crude
`peptides that contained deletion and/or addition com-
`Co-
`elution of the crude peptides with purified peptides (data
`not shown) suggested that peptides from Methods B
`and C coeluted with the purified peptides. On the other
`
`step. The standard protocols (Method zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`pounds as indicated by multiple peaks on HPLC. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`Method zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`A
`
`Peptide 4:
`
`Method B
`
`Method C
`
`Peptide 5:
`
`Peptide 6:
`
`FIGURE 2
`Reversed-phase HPLC of peptides prepared by multiple peptide synthesizer by different protocols. Column 1 shows peptides prepared by
`Method A where there was no mixing of resin. Column 2 shows peptides prepared by Method B in which mixing of the resin during cou-
`pling in DMF and deprotection was introduced. Column 3 shows peptides prepared by Method C in which mixing of the resin during coupling
`
`Fig. 2b.
`
`in mixed solvents. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`95
`
`MPI EXHIBIT 1037 PAGE 5
`
`MPI EXHIBIT 1037 PAGE 5
`
`

`

`T. Luu et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`such as peptides zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`when the ratio of DMF:DCM was 3:l. Peptide zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`intermolecular 8-sheet structure, thus making it difficult
`for the incoming amino acid to couple because of steric
`hindrance (12-14). Although this phenomenon
`is
`sequence-dependent, general solutions to this problem
`involve the use of more polar solvents and solvent ad-
`ditives such as HFIP to decrease the aggregation due
`to mostly 8-sheet formation (9, 15). However, these
`solvents may not be sufficient, and in fact may increase
`the aggregation primarily due to apolar side-chain pro-
`tecting groups such as tevt-butyl, BOC and trityl, thus
`decreasing coupling efficiencies. It has been reported
`that the aggregation of side-chain protecting groups
`leads to a collapsed gel structure, limiting further cou-
`pling (16). Therefore, a delicate balancing must be
`achieved between using a solvent system efficient enough
`to disrupt 8-sheet structures but does not interfere with
`side-chain protecting groups. The balance becomes
`more difficult to achieve when synthesizing several pep-
`tides simultaneously. For multiple peptide synthesis,
`the solvent system must be promiscuous and generally
`efficient for most of the sequences one commonly syn-
`thesizes. Several ways to achieve this balance such as
`the introduction of more polar side-chain protecting
`groups for individual amino acids or use of mixed sol-
`vent system have been discussed (17). The use of DCM
`to create mixed solvents has been suggested by Gause-
`pohl ei a/. for the peptide synthesis using multiple pep-
`tide synthesizer (3). In our search for a promiscuous
`solvent system we further explored the use of mixed
`solvents for multiple peptide synthesis and found that
`a solvent system containing 3 parts D M F and 1 part
`DCM was optimal. In this method the amino acids are
`dissolved in D M F and delivered to the reaction vessel
`containing the growing peptide chain assembly on resin,
`and then the appropriate amount of dichloromethane is
`added to achieve the 3:l ratio. This mixed solvent was
`able to yield pure peptides of 15 residues or longer,
`suggesting a general solution to 8-sheet formation and
`solvation of apolar side-chain protecting groups.
`In conclusion, the quality of crude peptides by mix-
`ing the resin during coupling increased 5-10-fold as
`compared to the quality of peptides produced by stan-
`dard methods without resin mixing during coupling and
`deprotection. The introduction of a mixed solvent sys-
`tem containing D M F and DCM had marginal effect on
`the quality of shorter pepetides, while it seemed to im-
`prove the the purity of longer peptides.
`
`al.
`
`hand, some peptides prepared by Method A had very
`little or no desired product, indicating the incomplete
`assembly of the peptide chain.
`The introduction of DCM in the coupling mixture
`has some effect on the purity of crude peptides. The
`effect was more prominent in cases of longer peptides,
`1-3. The amount of dichloromethane
`to be added was based on experiments in which the
`resin dispersion was evaluated at different ratios of
`D M F and DCM. The maximum dispersion was found
`1 con-
`tains four prolines. Unless care is taken to couple the
`prolines completely, synthesis of such peptides by Frnoc
`or Boc chemistry results in 20-30"" of desproline prod-
`ucts, whether the synthesis is automated or manual.
`Use of DCM seems to suppress the formation of these
`deletion structures.
`Simultaneous, multiple peptide synthesis using SPPS
`is challenging in terms of successful peptide assembly,
`cleavage and simultaneous isolation of 48 or more pep-
`tides. The peptide assembly step involves the activation
`of protected amino acid using reagents such as carbo-
`diimides, BOP, PyBOP, HBTU, CDI. and more re-
`cently by HOAt. These reagents render the carbonyl
`carbon of the carboxylic acid highly electrophillic and
`suitable for nucleophillic addition by the free amines on
`the resin. For successful peptide synthesis on solid
`phase the substitution reaction has to be complete,
`otherwise deletion pepetides are formed. PyBOP is a
`fairly good coupling agent when used in the presence of
`a tertiary base such as NMM or DIEA and produces
`less toxic by-products as compared to BOP (1 1). In our
`synthesis, we routinely used a double coupling strategy
`with 5 molar excess of activated amino acids over the
`amines on the resin to force the completion of amide
`bond formation. In the synthesis of one peptide at a
`time, resins can be checked for completion of coupling
`reactions by ninhydrin or fluorescamine tests. Simi-
`larly, tests for complete Fmoc deprotection can be per-
`formed by subjecting small amounts of resin to addi-
`tional deprotection and testing the supernatant for the
`presence of Fmoc by TLC or absorption at 290nm.
`However, such testing is practically impossible in si-
`multaneous synthesis of 48 or more peptides. Owing to
`the pseudodilution of reactive amines on the resin, ac-
`tivated amino acids are used in large excess over the
`nucleophiles available for reaction. The amino acid
`must be mixed thoroughly to increase the probability of
`amide bond formation. Therefore, changing the robot-
`ics to induce mixing of the resin and the activated amino
`acid solution resulted in higher quality of peptides with
`few deletion contaminants (Methods B and C).
`In solid-phase peptide synthesis, sufficient solvation
`of the growing peptide chain containing side-chain-
`protected amino acids is also absolutely necessary to
`achieve an efficient coupling reaction. If a sub-optimal
`solvent or solvent system is used for coupling reactions,
`the growing peptide chain assumes intramolecular or
`
`96
`
`ACKNOWLEDGEMENTS
`
`lye sincerel) thank Dr John Fara and Dr. Jeffery Winkelhake for
`their support and critical review of this manuscript.
`
`REFERENCES
`
`Ani. Chenz. Suc. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`I . hlerrifieid. R.B. (1963) J . zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`2. Groginskq, C. (1990) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`3. Gausepohl. H., Boulin, C . , Kraft, M. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`
`85, 2149-2154
`A m . Biurech. Lab. 8, 40-43
`& Frank, R.W. (1992)
`
`Pepride Res. 5. 315-320
`
`MPI EXHIBIT 1037 PAGE 6
`
`MPI EXHIBIT 1037 PAGE 6
`
`

`

`Automated multiple peptide synthesis
`
`Kopple, K.D., eds.) pp. 397-405, Pierce Chem Co., Rockford,
`IL
`
`& Dumias,
`
`J. Am. Chem. Soc. 108, 6493-6496
`15. Fields, C.G., Fields,G.B.,Noble, R.L.&Cross,T.A. (1989)lnt.
`J. Peptide Protein Res. 33, 298-303
`16. Atherton, E., Woolley, V. & Shepard, R.C. (1980) J. Chem. Soc.,
`Chem. Commun. 970-971
`17. Hyde, C., Johnson, T., Owen, D., Quibell, M. & Sheppard, R.C.
`lni. J. Peptide Protein Res. 43, 431-440
`
`A.G., Jelinski, L.W., Live, D., Kintanar, A. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`14. Ludwick, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
`J.J. (1986) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
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`
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`
`Shrikant Deshpande
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`97
`
`MPI EXHIBIT 1037 PAGE 7
`
`MPI EXHIBIT 1037 PAGE 7
`
`