Colony collections. Live queenright colonies and queenless sub-colonies, were collected from the Panama Canal region from 2015–2019 during May, June, and July. Atta sexdens males were collected immediately preceding a mating flight in May of 2019, then placed directly into vials of methanol. Collections were from: Barro Colorado Island (9.152208, -79.846485), Gamboa (9.119701, -79.6948), El Llano (9.282453, -78.9614), Plantation Road (9.076342005, -79.65978602), Creek Site on 16E (9.16313, -79.7449), Rio La Seda (9.1562, -79.73447), Fort Sherman (9.365336, 79.959310), Cardenas Creek (9.168533, 79.7519), and Parque Metropolitano (8.991493, -79.5442) (Table 1). Queenright colonies and queenless sub-colonies were maintained in a USDA containment facility (Permit P526P-19-02323) at Ohio State University in Columbus, Ohio, for up to four years before the collection of males.
Table 1 Collection information: Collection codes contain the collector’s initials, date of collection (year/month/day), and colony number; males were stored in MeOH as whole (W) or abdomen (A) samples
Genus
|
Species
|
Collection Code
|
Collection Location
|
Males in vial
|
Sample Type
|
Acromyrmex
|
echinatior
|
KAK190508-01
|
Gamboa
|
5
|
W
|
|
octospinosus
|
KAK190509-01
|
Gamboa
|
3
|
W
|
Apterostigma
|
dentigerum
|
JMJM170506-03
|
Plantation Rd
|
5
|
W
|
|
|
JMJM170511-01
|
16E
|
5
|
W
|
|
|
ARL190522-04
|
La Seda
|
4
|
W
|
|
|
RMMA190506-08
|
La Seda
|
6
|
W
|
Atta
|
cf. cephalotes
|
RMMA100615-01
|
Gamboa
|
1
|
W
|
|
sexdens
|
MRF190501-02
|
Gamboa
|
2
|
W
|
|
|
MRF190501-03
|
Gamboa
|
2
|
W
|
Cyphomyrmex
|
costatus
|
IMP170506-02
|
Plantation Rd
|
10
|
W
|
|
|
CTH170506-04 (1)
|
Plantation Rd
|
9
|
W
|
|
|
CTH170506-03
|
Plantation Rd
|
9
|
W
|
|
|
RMMA180620-01
|
La Seda
|
6
|
W
|
|
longiscapus
|
RMMA180623-05
|
El Llano
|
5
|
A
|
|
|
RMMA180623-13
|
El Llano
|
3
|
A
|
|
|
RMMA180622-03
|
16E
|
3
|
A
|
|
|
RMMA180622-09
|
16E
|
7
|
A
|
|
muelleri
|
NMH170515-02
|
16E
|
5
|
A
|
Sericomyrmex
|
amabilis
|
AC150523-01
|
16E
|
5
|
W
|
|
|
TK150507-06
|
La Seda
|
5
|
W
|
|
|
AMK150511-17
|
La Seda
|
5
|
W
|
|
|
MRB180715-01
|
Barro Colorado
|
8
|
A
|
|
opacus
|
RMMA150508-01
|
La Seda
|
9
|
A
|
|
|
MLD170522-01
|
Plantation Rd
|
10
|
A
|
|
|
MRB180725-04
|
Parque Metro
|
9
|
A
|
Mycetomollerius
|
opulentus
|
MRF190523-03
|
Fort Sherman
|
6
|
W
|
|
mikromelanos
|
CRC170508-01
|
La Seda
|
5
|
A
|
|
|
CRC170515-05
|
16E
|
4
|
A
|
|
|
CRC170513-10
|
La Seda
|
5
|
A
|
|
|
MRF190520-01
|
La Seda
|
5
|
W
|
|
|
RMMA190506-03
|
La Seda
|
6
|
W
|
|
zeteki
|
CRC170519-01
|
Cardenas Creek
|
6
|
W
|
|
|
ARL190522-07
|
La Seda
|
3
|
W
|
Paratrachymyrmex
|
bugnioni
|
APH150510-15
|
La Seda
|
5
|
W
|
|
cornetzi
|
MRB180728-01
|
Gamboa
|
10
|
A
|
|
|
MRF190522-05
|
La Seda
|
9
|
W
|
|
|
MRF190522-04
|
La Seda
|
4
|
W
|
Megalomyrmex
|
milenae
|
JMR190522-02
|
La Seda
|
5
|
W
|
Chemical Extraction. Live male ants (whole) were placed in vials with 40–100 µL of HPLC grade methanol (Table 1). Other males were trisected in a glass dish into head, thorax, and abdomen. The trisected parts were each placed in their own vial with methanol and passively extracted. The dish and tools used for trisection were rinsed in ethanol, methanol, and then pentane between trisections. See Table 1 for the extraction procedure used for each male collection.
Gas Chromatography-Mass Spectrometry. NMR spectra were obtained using a JEOL 400 YH NMR spectrometer. GC-MS was carried out in the EI mode using a Shimadzu QP-2010 GC-MS or a Shimadzu QP-2020 GC-MS equipped with an RTX-5, 30 m x 0.25 mm i.d. column. The instrument was programmed from 60° C to 250° C at 10°/min. HRMS were obtained by the Mass Spectrometry Laboratory of the School of Chemical Sciences at the University of Illinois Urbana-Champaign using a Waters Q-TOF Ultima ESI mass spectrometer or a Waters Micromass VG 70-VSE mass spectrometer. Previously identified tyramide compounds (1, 2, 3, 4, 6, 10, 11, 12, and 13) were identified through comparison to authentic samples (see Jones 2010; Adams 2010). Unidentified tyramides 5, 7, 8, and 9 were synthesized and identified by the methods described below.
Chemical Synthesis. N-[2-(4-Hydroxyphenyl)ethyl]-2-oxobutanamide (5). Compound 5 was prepared according to (Jones et al. 2010) using 0.21 g (2 mmol) of 2-keto-butyric acid and 0.137 g (1 mmol) of tyramine to provide 0.235 g of 4.1H NMR (400 MHz, CDCl3) δ 7.05 (1H, br s) 7.02 (2H, d, J = 4.2 Hz), 6.78 (2H, d, J = 4.2 Hz), 5.9 (1H br s), 3.51 (2H, q, J = 6.8 Hz), 2.93 (2H, q, J = 6.8 Hz), 2.76 (2H, t, J = 6.8 Hz), 1.07 (2H, t, J = 7.2 Hz), 13C NMR (100 MHz, CDCL3) δ 199.6, 160.3, 154.9, 129.9, 115.7, 40.9, 34.6, 30.4, 7.1; EIMS m/z 221 [M+] (1), 220(2), 164 (2), 121 (36), 120 (100), 107 (34), 103 (4), 93 (5), 91 (9), 77 (11), 57 (16), 51 (2), 41 (2); HRMS m/z 222.1134([M + 1]+), was calculated for C12H16NO3, 222.1130.
N-[2-(4-Hydroxyphenyl)ethyl]-2-hydroxybutanamide (8). A solution containing 20 mg of ketotyramide 5 in 3 mL of methanol was treated with three drops of 1 M NaOH and 60 mg of NaBH4 and stirred overnight at room temperature. The solvent was removed under reduced pressure and the residue was carefully acidified with 10% HCl and extracted twice with 5 mL portions of CH2Cl2. The combined CH2Cl2 extracts were dried over anhydrous MgSO4, and removal of the solvent provided hydroxy ketal 3 as a single component by GC/MS. 1H NMR (400 MHz, CDCl3) δ 1H NMR (400 MHz, D6-DMSO) δ 7.61 (1H, t, J = 8 Hz), 6.95 (2H, d, J = 8 Hz), 6.63 (2H, d, J = 8 Hz), 3.73 (1H, q, J = 4 Hz), 3.21 (2H, m, J = 8 Hz), 2.57 (2H, t, J = 8 Hz), 2.47 (1H, s), 2.26 (1H, s) 1.59 (1H, m), 1.43 (1H, m), 0.78(2H, t, J = 8 Hz). 13C NMR (100 MHz, CDCL3) δ 174.0, 156.1, 130.0, 126.0, 115.6, 72.5, 35.0 27.9, 9.9; EIMS m/z 223[M+] 223 (2), 194 (2), 164 (2), 121 (18), 120 (100), 107 (13), 91 (4), 77 (9), 65 (2), 59 (4); HRMS m/z 224.1291([M + 1]+), was calculated for C12H18NO3, 224.1287.
N-[2-(4-Hydroxyphenyl)ethyl]-2-methylpentanamide (7). A solution containing 0.25 g (2 mmol) of 2-methyl-butanoic acid in 20 mL of dichloromethane was treated with a slight excess of oxalyl chloride. After two hours, the solvent and excess oxalyl chloride were removed in vacuo. The residue was taken up in 10 mL of diethyl ether, and 0.27 g of tyramine (2 mmol) was added, followed by 15 mL of saturated NaHCO3. The biphasic mixture was stirred overnight. The aqueous layer was separated, and the organic phase was dried over MgSO4, filtered, and the solvent removed to provide 0.4 g of 7. 1H NMR (400MHz, CDCL3) δ 6.96 (2H, d, J = 8 Hz), 6.81 (2H, d, J = 8.4 Hz), 5.98 (1H, br s), 5.7 (1H, br s), 3.44 (2H, m), 2.69 (2H, t, J = 6.8 Hz), 2.12 (1H, sextet, J = 6.8 Hz), 1.54 (2H, m), 1.21 (2H, sextet, J = 7.2 Hz), 1.06 (3H, d, J = 3.2 Hz), 0.82 (3H, t, J = 7.2 Hz). 13C NMR (100 MHz, CDCL3) δ 177.9, 155.5, 129.8, 129.6, 115.8, 36.4, 34.8, 20.6, 17.9, 14.0; EIMS m/z 235 [M+] (1), 235 (1), 120 (100), 114 (23), 107 (13), 97 (4), 77 (4), 71 (4), 43 (14); HRMS m/z 236.1641([M + 1]+), was calculated for C14H22NO2, 236.165.
N-[2-(4-Hydroxyphenyl)ethyl]-2-methyl-( E,2 )-pentenamide (9). A solution containing 0.23 g (2 mmol) of E, 2-methyl-2-pentenoic acid in 20 mL of dichloromethane was treated with a slight excess of oxalyl chloride. After two hours the solvent and excess oxalyl chloride were removed in vacuo. The residue was taken up in 10 mL of diethyl ether, and 0.27 g of tyramine (2 mmol) was added, followed by 15 mL of saturated NaHCO3. The biphasic mixture was stirred overnight. The aqueous layer was separated and the organic phase was dried over MgSO4, filtered, and the solvent removed to provide 0.37 g of 8.1H NMR (400 MHz, CDCL3) δ 6.99 (2H, d, J = 8 Hz), 6.81 (2H, d, J = 8 Hz), 6.29 (1H, t, J = 7.2 Hz), 6.28 (2H, t, J = 7.2 Hz), 5.97 (1H, br-s), 3.51 (2H, m), 2.75 (2H, t, J = 6.7 Hz), 2.11 (2H, q, J = 7.2 Hz), 1.77 (3H, s), 0.98 (3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCL3) δ 170.2, 155.3, 138.8, 130.0, 129.8, 115.8, 41.4, 34.8, 21.7, 13.3, 12.6; EIMS m/z 233 [M+] (1), 233 (1), 120 (100), 114 (60), 107 (15), 97 (90), 77 (9), 69 (45), 41 (47); HRMS m/z 234.1484([M + 1]+), were calculated for C14H20NO2, 234.1494.