Cell Chemical Biology, Volume 26

Supplemental Information

C-Nucleoside Formation in the Biosynthesis

of the Antifungal Malayamycin A

Hui Hong, Markiyan Samborskyy, Yongjun Zhou, and Peter F. Leadlay

SUPPLEMENTARY FIGURES AND TABLES

C)

80 100 120 140 160 180 200 220 240 260 280 300 320 340m/z

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Relat

ive A

bund

ance 0

20

40

60

80

1000

20

40

60

80

100265.25

326.28 343.36189.28 235.29 293.29153.25113.30265.24

343.33326.29235.31189.32 293.29153.27113.33

311.33

221.31 251.29329.47155.33 286.39175.36

265.30

153.27146.35 282.33113.34 233.32205.35 247.34 300.38

221.26

251.27153.26 268.35132.29 185.35125.31

MS2: 343.2Malayamycin A std

Malayamycin A from S.chromofuscus WT

MS2: 343.2

MS2: 329.2

MS2: 300.2

MS2: 286.2

Desmethyl-Malayamycin Afrom ΔmalF

Descarbamoyl-Malayamycin Afrom ΔmalD

Desmethyl-descarbamoyl-Malayamycin A from ΔmalD

294.35

Figure S1. A) In-frame deletion of truD, malD, malF, and coaE genes in S. chromofuscus, Related toSTAR Method and Figure 3. Lane 1: marker; Lane 2 and 3: PCR product from truD (93 bp) and WT(1,897 bp), respectively; Lane 4: marker; Lane 5 and 6: PCR product from malD (2,437 bp) and WT(3,703 bp), respectively; Lane 7: marker; Lane 8 and 9: PCR product from malF (2,631 bp) and WT(3,315 bp), respectively; Lane10: marker; Lane 11 and 12: PCR product from coaE (1,108 bp) and WT(1,675 bp), respectively; Lane 13: marker. B) In-frame deletion of nikO gene in S. tendae, Related toSTAR Method and Figure 3. Lane 1: marker; Lane 2 and 3: PCR product from nikO (520 bp) and WT(1,942 bp), respectively. C) ESI-MS/MS spectra of malayamycin A and its analogous. MalayamycinAstandard ([M+H]+: 343.2), malayamycin A produced from S. chromofuscus WT, desmethyl-malayamycinA from ΔmalF ([M+H]+: 329.2), descarbamoyl-malayamycin A from ΔmalD ([M+H]+: 300.2) anddescarbamoyl-descarbamoyl-malayamycin A from ΔmalD ([M+H]+: 286.2).

Lane(kb)

1 2 3 4 5 6 7 8 9 1011 12 13

108.06.05.04.03.02.01.51.0

0.5

A) B)Lane(kb)20107.05.04.03.02.01.51.00.70.50.40.30.2

1 2 3

TruD_SS --------------MTGTLVLKHRQEDFRVRENLVVALTDAAAATHRYLLLHKRGHTTME 46TruD_SM ---------------VEYPVLKAHDTDFVVQESMVLPAQDDPDAPYQYLRLRKRGYTTFE 45TruD_SC ---------------VEYPILKAHDTDFVVQESMVLPVQDDQDAPFQYLRLRKRGYTTFE 45TruD_HP MNLNFMPLLHAYNHASIDFHFNSSARDFCVHEVPLYEFSNT--GEHAVIQVRKSGLSTLE 58TruD_AF MEEKVGIECYITSTPGMGGEIKAEPEDFYVEEIAEFNLSDE--GDFLIIRVEKKNWDTLN 58TruD_EC -MIEFDNLTYLHGKPQGTGLLKANPEDFVVVEDLGFEPDGE--GEHILVRILKTGCNTRF 57

TruD_SS AVRLVADRLGVAREDVGYAGLKDEDGITEQLLSVPLASLAA--DALPAAGLVEGAGPERM 104TruD_SM ALERIAALCGVSASDLSTAGLKDEDAVTEQHIACRG-RLPR--DAITEFNARHA-SGART 101TruD_SC AVERIAAVCGVPASDLSTAGLKDEDAVTEQHLACRG-GLSP--DGIAEFNTRHG-SGERT 101TruD_HP MLQIFSQILGVRIAELGYAGLKDKNALTTQFISLPKKYAPLLEKNTSNFQE-------KN 111TruD_AF FARVLSNALGISQKRISFAGTKDKRALTVQYFSIYGVKKE----EIERVNL-------KD 107TruD_EC VADALAKFLKIHAREVSFAGQKDKHAVTEQWLCARVPGKEM--PDLSAFQL-------EG 108

*TruD_SS LSLSHYGFGREPLTVGQLNGNGFRVVLRDLDEAAAAR--LVDRQRINLLFVNYYDTQRFG 162TruD_SM MTLTTHGYGHLNLRAGQLEGNAFRITVRGLSSGFATTLGALGERAENLFFVNYYDVQRFG 161TruD_SC MRLSLHGYGHQGLRAGHLEGNGFRIVVRGLSAGFVRTVEALGERAGDLFFVNYYDTQRFG 161TruD_HP LKILSLNYHHNKIKLGHLKGNRFFMRFKKMTPLNAQKTKQVLEQIAQFGMPNYFGSQRFG 171TruD_AF AKIEVIGYARRAIQLGDLLGNFFRIRVYGCR--DGEIFQETRNELMEKGTPNFFGLQRFG 165TruD_EC CQVLEYARHKRKLRLGALKGNAFTLVLREVS--NRDDVEQRLIDICVKGVPNYFGAQRFG 166

TruD_SS VPGGPKRTHLVGEALLKEDWALARQEL-------AGLGAPESGEAA-------------- 201TruD_SM VAGGPRTTHQIGGALLEEDYGTALALV-------RESQSPEADHAR-------------- 200TruD_SC VAGGPRTTHQIGRALLEEDHGTALGLV-------RESKSPEAERAR-------------- 200TruD_HP KFNDNH--QEGLK-ILQNQTKFAHQKLNAFLISSYQSYLFNALLSKRLEISKIISA---- 224TruD_AF SIRFIT--HEVGK------LILQNNYEEAFWVYVAKPFEGENEEVRKIRE--ILWETRDA 215TruD_EC IGGSNL--QGAQR-WAQTNTPVRDRNKRSFWLSAARSALFNQIVAERLKKADVNQVVDGD 223

TruD_SS --------RWTG-------------QDRALFRALDPRTVAFYLAAYSSYDWNARVRDLIG 240TruD_SM --------RFTG-------------PAAQFFDRIDPRVRAFYLCARASWVWNGQLAALVK 239TruD_SC --------LFTG-------------SAAAFFDRIDPRVRAFYLCAHASYVWNAQLAALLR 239TruD_HP -FSVKENLEFFKQKN--------LSVDSDTLKTL-------------------------K 250TruD_AF KLGLRELPKYLRYERNLLQKLREGKSEEEALLSLPKNLKMMFVHAYQSYIFNRLLSERIR 275TruD_EC ALQLAGRGSWFVATTEELAELQRRVNDKE----------LMITA-------------ALP 260

TruD_SS SLCPDDAPESAVDGLPYRFPT--TA------------------EGVAALLAACHELPYTR 280TruD_SM KVSHHPLDETLREGLPYVFTT--HR------------------DDVLALLRHATSLPYER 279TruD_SC KVGRHPLDETVREGIPYTFAT--HR------------------DDVLALLQEATTLPYER 279TruD_HP NQAHPFKILEGDVMCHYPYG---KFFD--ALELEKEGERFLKKEVAPTGLLDGKKALYAK 305TruD_AF QFGSLKTLEEGDFACYLTFKTRPTFSDCSEVEVNEARVRFLVKERVASL--ALPLVGYDT 333TruD_EC --------GSGEWGT---QREALAFEQA-AVAAETELQALLVREKVEAA--RRAMLLYPQ 306

TruD_SS Y-AYRDGPVERP-TTRPTVIQTAITVGADGPD---------------------------- 310TruD_SM Y-RWSNAAIRRTQGLRRTVVQTRIRVDRTSED---------------------------- 310TruD_SC H-RWDGGEMRRTQGLRPTVVQTRVRVDRTGED---------------------------- 310TruD_HP NLSLE--------------IEKEFQHNLLSSHAKTL----------GSRRFFWVFVENVT 341TruD_AF K--LK--------------GWSRIALDFLSEDNLDLSSFKTKHKEFSSSGSY-RPADTLI 376TruD_EC QLSWN--------------WWD-------------------------------------- 314

TruD_SS -DAFPGRRAVEVSFLLPSGCYATAALRQLVLRR-----------342TruD_SM -SDTPGRYACEIALFLPPGCYATNAVSQWVAWIARTDAPRNANA353TruD_SC -TDTPGRHACELTLFLPSGCYATNAVAQWVAWAG----------343TruD_HP SQYVKEKAQFELGFYLPKGSYASALLKEIKHEKGENNDEF----381TruD_AF EHTGLSFTDSTFSFYLPRGCYATVFLREFLKTELS---------411TruD_EC ------DVTVEIRFWLPAGSFATSVVRELINTTGDYAHIAE---349

Figure S2. Sequence alignment of TruD family of tRNA pseudouridine synthases, Related to Figure 5.The sequences of TruDs [TruD_SM: TruD from Streptomyces malaysiensis; TruD_SC: TruD fromStreptomyces chromofuscus; TruD_SS: TruD from Streptomyces sp. ID38640 (accession no. AVT42379);TruD_HP : TruD from Helicobacter pylori (accession no. NP_207718); TruD_AF: TruD fromArchaeoglobus fulgidus (accession no. O28596); TruD_EC: TruD from E. coli (accession no. AQZ29382)]are aligned using Clustal Omega. The conserved sequence motifs are highlighted in boxes. The catalyticaspartate is denoted with an asterisk.

Figure S3. Phylogenetic analysis of YeiN-related glycosidase and putative glycosynthase enzymes,Related to Figure 4. Sc: Streptomyces chromofuscus; St: Streptomyces tendae; Sm: Streptomycesmalaysiensis; ShYeiN: YeiN from Streptomyces himastatinicus (accession no. WP_009720488); EcYeiN:YeiN from Escherichia coli (accession no. AAA60517); AlnA: alnumycin biosynthesis from Streptomycessp. CM020 (accession no. ACI88875); SaInda: indigoidine biosynthesis from Streptomyces albus(accession no. AMM12432); Sdma: showdomycin biosynthesis from Streptomyces showdoensis ATCC15227.

Figure S4. Comparison of ESI-MS/MS spectra of 3'-EPUMP ([M+H]+: 395.2) and 3'-EP-Ψ-MP([M+H]+: 395.2), Related to Figure 4. Loss of C-N linkage in 3'-EP-Ψ-MP abolished the fragment at m/z283.2, which was a key fragment in the MS/MS of 3'-EPUMP.

A)

B)

Figure S5. Analysis of N-malayamycin A by MS and 1H NMR, Related to Figure 5A. A) High-resolution UPLC-MS analysis of N-malayamycin A and malayamycin A from fermentation extract of S.chromofuscus ΔtruD::nikO mutant. B) Comparison of 1H spectra of N-malayamycin A purified fromfermentation of S. chromofuscus ΔtruD::nikO mutant and from chemical synthesis (Hanessian et al., 2005).

[M+H]+: 343.1248(calculated for C13H19N4O7)

C)

N

HO

OH

O

HN

H2N

O

O

HO HO

HN

N

O

O

OH

H

496.1674

H2N

O

O

HO HO

HN

N

O

O

OH

H

288.0826

H2N

O

O

HO HOOH

H

176.0553

a ba

b

Nikkomycin Z

N

HO

OH

O

HN

H2N

O

O

HO HOOH

NHHN

O

O

H2N

O

O

HO HOOH

H

H

496.1674

NH

HN

O

O

288.0826

H2N

O

O

OH

NH

HN

O

O

H

H2N O

NH

HN

O

O

H

208.0717

aa

-2 H2O

252.0615

- CO2

pseudo-Nikkomycin Z

Figure S6. MS/MS and MS3 analysis of nikkomycin Z and Ψ-nikkomycin Z, Related to Figure 5B. A)MS/MS and MS3 spectra of nikkomycin Z. B) MS/MS and MS3 spectra of Ψ-nikkomycin Z. C)Fragmentation pathways for the generation of m/z 176 fragment in nikkomycin Z and of m/z 208 fragmentin Ψ-nikkomycin Z.

Figure S7. Comparison of Ψ–nikkomycin Z production with and without uracil feeding to theΔnikO::malO_Sm mutant, Related to Figure 5B. When uracil (0.4%) was fed to the ΔnikO::malO_Smmutant, the production of -nikkomycin Z was increased 5-fold.

0 5 10 15Time (min)

Ψ-Nikkomycin Z

Nikkomycin Z

ΔnikO::malO_Sm

ΔnikO::malO_Sm+ uracil

Table S1. Deduced Functions of ORFs in the mal Biosynthetic Gene Cluster of Streptomyces

malaysiensis DSM 14702, Related to Table 1.ORFa Sizeb Protein Homolog (Accession No.), Origin I/Sc

(%)Proposed Function

0224c 246 ribonuclease PH (WP_047176847)Streptomyces sp. MNU77

93/95 unknown function

0225coaE

206 dephospho-CoA kinase (ACU97583)Saccharomonospora viridis

40/50 unknown function

0226ctruD

353 tRNA -uridine synthase D (AGZ78434)Streptomyces chromofuscus

98/99 provision of -UMP

0227cmalA

513 major facilitator transporter (AEZ64581)Streptomyces chromofuscus

97/98 transporter

0228cmalM

245 putative hydroxylase (AEZ64582)Streptomyces chromofuscus

76/81 hydroxylase

0229cmalO

479 UDP-GlcNac 1-carboxy-vinyl transferase(AEZ64583) Streptomyces chromofuscus

82/87 enoylpyruvyl-UMPsynthase

0230cmalI

208 nikkomycin biosynthesis protein NikI(AEZ64584) Streptomyces chromofuscus

83/90 hydroxylase

0231cmalB

263 short chain reductase (AEZ64585)Streptomyces chromofuscus

83/86 unknown function

0232cmalL

246 protein tyrosine/serine phosphatase(ABV97039) Streptomyces chromofuscus

72/81 octosyl acid phosphatehydrolase

0234cmalC

257 no hit unknown function

0237malJ

454 radical SAM protein (ABX24496)Streptomyces chromofuscus

83/87 octosyl acid phosphatesynthase

0238malK

395 aminotransferase NikK (AEZ64589)Streptomyces chromofuscus

84/89 aminotransferase inAHA synthesis

0239malD

490 carbamoyltransferase (AEZ64590)Streptomyces chromofuscus

92/95 carbamoyl transferase

0240 276 DNA topology modulation protein(AEZ64591) Streptomyces chromofuscus

64/69 unknown function

0241malE

370 O-methyltransferase (AEZ64592)Streptomyces chromofuscus

81/87 methyltransferase

0242malR1

308 transcriptional regulator ArsR(NP_033310326) Streptomyces iakyrus

45/57 regulator

0243 473 peptidase M20 (ABL83816) Nocardiodessp. JS614

76/84 unknown function

aSuffix c denotes complementary strandbAmino acidscIdentity/similarity

Table S2. Plasmids used in this work, Related to STAR MethodsPlasmid Genotype/Characteristics Reference

pYH7

E.coli-Streptomyces shuttle vector

Sun et al.,

2010

pYH7-truD truD gene (from S. chromofuscus) disruption construct inwhich a 966 bp internal fragment of truD was deleted in-frame

this work

pYH7-malF malF gene (from S. chromofuscus) disruption construct inwhich a 684 bp internal fragment of malF was deleted in-frame

this work

pYH7-malD malD gene (from S. chromofuscus) disruption construct inwhich a 1266 bp internal fragment of malD was deleted in-frame

this work

pYH7-coaE coaE gene (from S. chromofuscus) disruption construct inwhich a 567 bp internal fragment of coaE was deleted in-frame

this work

pYH7-nikO nikO gene (from S. tendae) disruption construct in which a1422 bp internal fragment of nikO was deleted in-frame

this work

pGP9E.coli-Streptomyces shuttle vector, attP (ΦBT1),int, PactI

Gregory et

al., 2003

pGP9-truD pseudouridine synthase truD (from S. chromofuscus)complementation plasmid

this work

pGP9-nikO enoylpyruvyl-UMP synthase nikO (from S. tendae)complementation plasmid

this work

pGP9-malD carbamoyltransferase malD (from S. chromofuscus)complementation plasmid

this work

pIB139 E.coli-Streptomyces shuttle vector, attP (ΦC31),int, PermE*

pIB139-truD_Sm pseudouridine synthase truD (from S. malaysiensis)complementation plasmid

this work

pIB139-nikO enoylpyruvyl-UMP synthase nikO (from S. tendae)complementation plasmid

this work

pIB139-malO_Sm enoylpyruvyl-UMP synthase malO (from S. malaysiensis)complementation plasmid

this work

pIB139-malO_Sm-truD_Sm

enoylpyruvyl-UMP synthase malO (from S. malaysiensis)together with pseudouridine synthase truD from (S.malaysiensis) complementation plasmid

this work

pET28a(+) E. coli protein expression vector Invitrogen

pET28a-truD_Sm truD (from S. malaysinesis) protein expression constructwith N-terminal His-tag based on pET28a(+)

this work

pET28a-YeiN E. coli pseudouridine-5′-phosphate glycosidase yeiN proteinexpression construct with N-terminal His-tag based onpET28a(+)

this work

pET28a-malO_Sm malO (from S. malaysinesis) protein expression constructwith N-terminal His-tag based on pET28a(+)

this work

pET28a-nikO nikO (from S. tendae) protein expression construct with N-terminal His-tag based on pET28a(+)

this work

Table S3. Oligonucleotide primers used in this work, Related to STAR MethodsPrimer Nucleotide sequence (5' to 3')

primers for protein expressionTruD_Sm-p28_Fd CTGGTGCCGCGCGGCAGCCATATGGAGTACCCCGTCCTCAAGGCCCACTruD_Sm-p28_Rv TCCACCAGTCATGCTAGCCATATGCTAGGCGTTGGCGTTCCG GGGTGCMalO_Sm-p28_Fd CTGGTGCCGCGCGGCAGCCATATGGAACCTCGGCTGACCCCCTCCGTG

MalO_Sm-p28_Rv TCCACCAGTCATGCTAGCCATATGTTACTCCTGGGA GCCCGG TGGGTCNikO-p28_Fd CTGGTGCCGCGCGGCAGCCATATGCAGGACCGTTGGCGAGAACCCGCTNikO-p28_Rv TCCACCAGTCATGCTAGCCATATGTCAGGCCGGGTCCACACCGGCCCCYeiN-p28_Fd CTGGTGCCGCGCGGCAGCCATATGTCTGAATTAAAAATTTCCCCTYeiN-p28_Rv TCCACCAGTCATGCTAGCCATATGTTAACCCACGAGACGCTGATATTCprimers for genes in-frame deletion based on pYH7 vectortruD-L1 TGATCAAGGCGAATACTTCATATGTCTGGTACTTCAACTCCGAGTGGCtruD-L2 GGGCAGGAAGAGGGTCACTCCGTTCCTTTCGTCCTGtruD-R1 AACGGAGTGACCCTCTTCCTGCCCTCCGGCTGCTACtruD-R2 CCGCGCGGTCGATCCCCGCATATGAGATCGCGAGCAGCAGACAGACGAcoaE-L1 TGATCAAGGCGAATACTTCATATGAGCGCATGAGGGTGTTGAAGCTGTcoaE-L2 CATCGTGGCGGAGTCCTTCGGCATCCGGCGATTCTAcoaE-R1 ATGCCGAAGGACTCCGCCACGATGACGGTCAGGACCcoaE-R2 CCGCGCGGTCGATCCCCGCATATGATCCTCTGGTGGACCCGTGGCTCTmalD_L1 GTCCACCGGGACTGATCAAGGCGAATACTTCATTCGAGTACGTCCACGAACTCmalD_L2 GAGCGGTACAGCAGACGCAAGAACAACACCAGTCTCAACATCAAGGmalD_R1 CCTTGATGTTGAGACTGGTGTTGTTCTTGCGTCTGCTGTACCGCTCmalD_R2 CTTCCCCGTCCGGGACCCGCGCGGTCGATCCCCGCAGTGGAAATGTGCGACmalF_L1 GTCCACCGGGACTGATCAAGGCGAATACTTCAACGAGCTGAGGAAGAAGAAGmalF_L2 CTCACCTGCCGGGACATCTTCACCTCGCACGATCTGCGCATGCTGmalF_R1 CAGCATGCGCAGATCGTGCGAGGTGAAGATGTCCCGGCAGGTGAGmalF_R2 CTTCCCCGTCCGGGACCCGCGCGGTCGATCCCCGCAATCTGGTCTTCTCnikO_L1 TGATCAAGGCGAATACTTCATATGACCCACCTTTTCTACTTCAACGACnikO_L2 AGGCCCAGGACGGGTGGTACCTCCTGAGCTGAGTGGnikO_R1 GGAGGTACCACCCGTCCTGGGCCTCGACCGCGACGAnikO_R2 CCGCGCGGTCGATCCCCGCATATGCGGTTGTATTCGTCCTGCACCACTprimers for PCR screening of deletion mutantstruD-CP1 TGTCATACTTCTCCGTCGTCCTGtruD-CP2 ACCTACGCCGATGTCAAGGATCCcoaE-CP1 TACTGGACCGCAGTGCCTACATCcoaE-CP2 TGTTCTTCGTCAACTACTACGACmalD-CP1 TTGATGGCAGAAGGCGGTCTTGmalD-CP2 TTCTACGCCAAGGACGAGCACmalF-CP1 TTTCGTGAGGACGACCTGGATCATGmalF-CP2 ACGTCCCTATGGAGGAGCACATGACnikO-CP1 ACACGTTCGTCTGGCTCGACCTGCnikO-CP2 TGCGCGGTCGATGAGCTGGTCGTAprimers for genes complementation using pGP9 vectortruD-p9_Fd GATTAATTAAGGAGGACA CATATG GAGTACCCCATCCTCAAGGCTCACtruD-p9_Rv GGCTGGGCAGAATAGGGC CATATG TCAGCGCTCCGACCCGGCCCAGGCmalD-p9_Fd GATTAATTAAGGAGGACA CATATG GCGCACATCCTCGGCCTGTCCTGCmalD-p9_Rv GGCTGGGCAGAATAGGGC CATATG TCACAGGACGGGGATGCCATGGAGnikO-p9_Fd GATTAATTAAGGAGGACA CATATG CAGGACCGTTGGCGAGAACCCGCTnikO-p9_Rv GGCTGGGCAGAATAGGGC CATATG TCAGGCCGGGTCCACACCGGCCCCprimers for genes complementation using pIB139 vectortruD_Sm-p139_Fd GCCGGT TGGTAG GATCCA CATATG GAGTACCCCGTCCTCAAGGCCCAC

truD_Sm-p139_Rv CCTCTA GAGGAT CCCCAA CATATG CTAGGCGTTGGCGTTCCGGGGTGCnikO-p139_Fd GCCGGT TGGTAG GATCCA CATATG CAGGACCGTTGGCGAGAACCCGCTnikO-p139_Rv CCTCTA GAGGAT CCCCAA CATATG TCAGGCCGGGTCCACACCGGCCCCmalO_Sm-p139_Fd GCCGGT TGGTAG GATCCA CATATG GAACCTCGGCTGACCCCCTCCGTGmalO_Sm-p139_Rv CCTCTA GAGGAT CCCCAA CATATG TTACTCCTGGGAGCCCGGTGGGTCtruD_Sm-XbaI_Fd GAGTAACATATGTTGGGGATCCTCTAGAgaaggaacggagtgaacATGGAGTACCCtruD_Sm-XbaI_Rv GCGCGCGGCCGCGGATCCTCTAGACTAGGCGTTGGCGTTCCGGGGTGC

Table S4. NMR spectral data of 3'-enolpyruvyl-pseudouridine monophosphate (3'-EP-Ψ-MP)and 3'-enolpyruvyl-uridine monophosphate (3'-EPUMP) in [2H6]DMSO, Related to Figure 4.

3’-EP-Ψ-MPa 3’-EPUMPb

Position δC δH (multi, J in Hz) δC δH

C-2 151.3 151.7C-4 163.7 164.0C-5 110.3 103.2 5.67C-6 140.5 7.57 (1H, s) 7.74 (1H, d)C-1’ 78.4 4.60 (1H, d, 5.35) 88.5 5.88C-2’ 72.0 4.30 (1H, m) 72.0 4.42C-3’ 77.8 4.36 (1H, m) 77.1 4.5C-4’ 79.1 4.05 (1H, m) 80.9 4.20C-5’a 64.3 3.92 (1H, m) 65.7 4.06C-5’b 3.80 (1H, m) 3.99=CH2a 95.9 5.21 (1H, s) 97.5 5.33=CH2b 4.81 (1H, s) 4.88C= 151.0 150.9COOH 164.2 164.6

aMeasured at 500 MHz (1H) and 125.7 MHz (13C);bData from (Ginj et al., 2005). Measured at 500 MHz (1H) and 125.9 MHz (13C).